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WO1997015588A1 - Proteine/cathepsine a protectrice et precurseur: cristallisation, diffraction des rayons x, determination de structure tridimensionnelle et elaboration rationnelle de substances therapeutiques - Google Patents

Proteine/cathepsine a protectrice et precurseur: cristallisation, diffraction des rayons x, determination de structure tridimensionnelle et elaboration rationnelle de substances therapeutiques Download PDF

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Publication number
WO1997015588A1
WO1997015588A1 PCT/US1996/017325 US9617325W WO9715588A1 WO 1997015588 A1 WO1997015588 A1 WO 1997015588A1 US 9617325 W US9617325 W US 9617325W WO 9715588 A1 WO9715588 A1 WO 9715588A1
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ppca
pppca
data
atomic
model
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PCT/US1996/017325
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WO1997015588A9 (fr
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Gabrielle Rudenko
Alessandra D'azzo
Wim G. J. Hol
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St. Jude Children's Research Hospital
University Of Washington
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Priority to AU11157/97A priority Critical patent/AU1115797A/en
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Publication of WO1997015588A9 publication Critical patent/WO1997015588A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present invention is in the fields of molecular biology, protein purification, protein crystallization, x-ray diffraction analysis, three-dimensional structure determination and rational drug design (RDD)
  • the present invention provides crystallized protective protein/cathepsin A (PPCA) and its precursor (pPPCA)
  • PPCA crystallized protective protein/cathepsin A
  • pPPCA precursor
  • the crystallized PPCA or pPPCA is analyzed by x-ray diffraction techniques
  • the resulting x-ray diffraction patterns are of sufficiently high resolution to be useful for determining the three-dimensional structure of the PPCA or pPPCA protein, and for RDD Related Background A rt
  • the human protective protein/cathepsin A (PPCA, also known as human protective protein or HPP) has been identified as the primary genetic defect underlying galactosia dosis (d'Azzo et al , Proc Natl Acad Sci US A 79 4535- 4539 ( 1982)), a Ivsosomal storage disease inherited as an autosomal recessive trait Patients with this disorder are diagnosed as having drastically reduced ⁇ -galactosidase and neuraminidase activities in their cell lysosomes Examples of Ivsosomal storage diseases are presented in Table 316- 1 of Braun wald et al eds Harrison s Principles of Internal Medicine 1 Ith Ed , pp 1661 -1671.
  • Lysosomal PPCA has cathepsin A/deamidase/esterase activities which are exerted in vitro on a specific subset of bioactive peptides
  • Non-limiting examples of those hydrolyzed by PPCA are substance P and substance P-free acid. oxvtocin and oxytocin-free acid, neurokinin A, angiotensm I.
  • bradykinin (Jackman infra (1990) Fur t hermore the enzyme inactivates endothelin I activity in rat smooth muscle cells and normal human tissues This activity was deficient in liver from a galactosiahdosis patient (lto infra 1995. Jackman et al J Biol Chem 26 7 2872-2875 , ( 1992) Endothelms (ET-1. ET-2 and ET-3) are potent vasoconstrictors and elevate blood pressure in mammals The ⁇ also influence cell proliferation and hormone production and have been implicated in cardiovascular disorders ranging from hypertension to stroke to ischemic heart disease (Rubanyi and Polokoff Pharmc Rev 46 325-415 (1 94))
  • the present invention provides methods of expressing, purifying and crystallizing a human protective protein/cathepsin A (PPCA) and its precursor, precursor protective protein/cathepsin A (pPPCA)
  • PPCA human protective protein/cathepsin A
  • pPPCA precursor protective protein/cathepsin A
  • the present invention also provides methods for obtaining crystallized PPCA or pPPCA that can be analyzed to obtain x-ray diffraction patterns of sufficiently high resolution to be useful for three-dimensional structure determination of the protein
  • the x-ray diffraction patterns can be either analyzed directly to provide the three dimensional structure (if of sufficiently by high resolution), or atomic coordinates for the crystallized PPCA or pPPCA, as provided herein, can be used for structure determination
  • the x-ray pattem/diffraction patterns obtained by methods of the present invention, and provided on computer readable media, are used to provide electron density maps
  • the ammo acid sequence is also useful for three-dimensional structure determination
  • the data is then used in combination with phase determination (e g using multiple isomorphous replacement (MIR) molecular replacement techniques) to generate electron density maps of a PPCA or a pPPCA, using a suitable computer system
  • the electron density maps provided by analysis of either the x-ray diffraction patterns or working backwards from the atomic coordinates, provided herein, are then fitted using suitable computer algorithms to generate secondary, tertiary and/or quaternary domains of a PPCA or a pPPCA, which domains are then used to provide an overall three- dimensional structure, as well as expected binding and active sites of the PPCA or pPPCA pPPCA has some of the active and binding sites of PPCA . except for changes in structure due to the presence of the portion of the pPPCA which is deleted during maturation to PPCA (e g , residues 285-298 of Figure 13)
  • RDD rational drug design
  • PPCA-specific structural feature or biological activity preferably as associated with a PPCA- or pPPCA-related pathology, e g , protective activity (e g , modulation of ⁇ -galactosidase activity and neuraminidase (NA) activity), and peptide or enzyme modulating activity (eg , of endothelin I (serine carboxypeptidase), neuropeptides, cathepsin A, and the like), according to known assays
  • resulting ligands provided by methods of the present invention are synthesized and are useful for treating, inhibiting or preventing at least one of PPCA-specific structural feature or biological activity, preferably as associated with a PPCA- or pPPCA-related pathology, e g , protective activity (e g , modulation of ⁇ -galactosidase activity and neuraminidase (NA) activity), and peptide or enzyme modulating activity (eg ,
  • Figure 1 is a schematic ribbon diagram of the PPCA monomer (monomer 1 ), where Secondary structure assignments are according to DSSP ( absch and Sander, Biopolymers 22.2571-2637 (1983))
  • the 'core' domain is shown in yellow
  • the 'cap' domain consists of a 'helical' subdomain, in red, and a 'maturation' subdomain, in orange
  • FIG. 2 is stereo diagram is presented of the C ⁇ trace of the PPCA monomer 1 with numbering of selected residues
  • the residues forming the ⁇ -helices and ⁇ -strands are as follows according to DSSP
  • Figure 5 is a schematic ribbon diagram is presented of the PPCA dimer viewed approximately along the two ⁇ fold axis
  • the core domain is yellow while the cap domain consists of a helical subdomain in red and a maturation subdomain in orange
  • the core domain is green, while the cap domain consists of a blue helical subdomain and a light blue maturation subdomain
  • Figure 6A-B is a representation of the molecular surface of the PPCA dimer The surface was calculated with GRASP (Nicholls.
  • Figure 7A-F presents a topological comparison of 6 members of the hydrolase fold family
  • the arrangement of structural elements in the central core domain (in green and yellow) of the different proteins is generally similar
  • the cap domains (in red) vary greatly
  • Figure 7A shows the PPCA precursor cap domain that consists of two subdomams one ⁇ -helical and the other mainly ⁇ -sheet
  • Figure 7B shows CPW (3SC2, Liao et al (1992) infra), cap domain helical
  • Figure 7C shows CPY (LYSC, Endnzzi et al ( 1994), infra)
  • Figure 7D shows dehalogenase (2HAD, Franken et al J EMBO 10 1297-1302 (1991 )), cap domain helical but quite different from the serine carboxypeptidases
  • Figure 7E shows lipase from Pseudomonas glumae (
  • Figure 8A-B shows the superposition of the C traces from the PPCA and CPW monomers, showing that the major differences between the two enzymes are localized in the cap domain PPCA has a large 'maturation' subdomain and the 'helical subdomain is rotated with respect to the CPW counterpart ( Figure drawn with the O program (Jones ( 1991 ), infra))
  • Figure 8B shows the C traces from the PPCA and CPW dimers after the core domains from the subunits (shown on the right hand side of the two dimers) have been superimposed Notice the remarkable difference in mutual orientation (of 15°) of the two subunits on the left hand side of the two dimers which has been accentuated by an arrow ( Figure drawn with the O computer program (Jones ( 1991 ), supra))
  • Figure 9 is a stereo view of the Ca trace of PPCA monomer 1 highlighting regions involved in the maturation event Color scheme for the trace is as follows core domain in light blue helical subdomain in red maturation subdomain in orange with the exception of the excision peptide (residues 285-298) which is shown in blue Orange sphere mark the residues 272 and 277 marking the beginning and end of the blocking peptide
  • the catalvtic triad Ser 150 His 429 and Asp 372 is shown as light blue spheres
  • Two cvsteines Cys 253 and C>s 303 referred to in the discussion are colored green (This Figure generated using MOLSCRIPT (Kraulis ( 1991 ) infra))
  • Figure 10 is a close-up representation of the blocking peptide (residues 272-277) bound in the active site rendering the catalvtic triad solvent inaccessible Residues from the maturation subdomain are shown in orange residues fro the helical domain in magenta and residues from the core domain in cyan. The excision peptide is shown in blue. Side chains are shown for residues making extensive contacts with the blocking peptide or if mentioned in the text. The catalytic triad is shown in white. ( Figure drawn with O (Jones (1991), infra)).
  • Figure 11 is a representation of elements proposed to be involved in the activation mechanism of the precursor form of PPCA as discussed in the text.
  • the C-trace of the core domain is shown in cyan, the helical subdomain in red, the maturation subdomain in orange, and the excision peptide is shown in blue. Relevant side chains are depicted and labeled. Rearrangement of the residues 254-302 limited by the disulfide Cys 253 and Cys 303 would free up the active site cleft.
  • a charge cluster Arg 262, Glu 264, Arg 298 and Asp 300 occupies a strategic position within the maturation subdomain, possibly involved in pH dependent regulation of conformational changes.
  • BIOGRAF BIOGRAF Construct Users Guide Version 3.2.1 , June 1993.
  • Figure 12 is a schematic representation of the proposed activation of PPCA.
  • the active site cleft is formed by the core domain (indicated as 'core' in the above scheme) and the helical subdomain (indicated as ' ⁇ ').
  • the maturation subdomain (indicated as 'm') contains the residues that block the active site cleft rendering the precursor enzymatically inactive, shown in structure 1.
  • the precursor undergoes activation.
  • conformational rearrangements induced by low pH might render the excision peptide more accessible to proteases as a first step, followed by cleavage of the polypeptide chain removing the excision peptide.
  • FIG. 13 shows the amino acid sequence of a human pPPCA.
  • the underlined portion shows an excision peptide for conversion to the mature form, PPCA.
  • Figure 14 shows the amino acid sequence of a human PPCA.
  • Figure 15 shows a sequence alignment between pPPCA, CPW and CPY (top three sequences shown). Identical residues among all three sequences are boxed. Residue numbering is included for the pPPCA amino acid sequence.
  • the alignment was made using the GCG program PILEUP (GCG version 8), then manually adjusted using 3D-structural knowledge from the superposition of the CPW (Liao et al. , 1992) and CPY (Endrizzi et al. , 1994) atomic coordinates. The alignment was later used to design a multi-Ala search probe for molecular replacement calculations shown in the fourth sequence shown as 'model'.
  • pPPCA protein can be divided in two domains: a 'core' domain (residues 1-182 and 303-452) and 'cap' domain (residues 183-302).
  • the secondary structure elements for the PPCA precursor are depicted with shaded bars (for details on the assignment and nomenclature, see Rudenko et al. Structure 3:1249-1259 (1988) ).
  • Figure 16 shows a schematic representation of a 'bootstrapping' cycle as described in Example 2.
  • Figure 17 is a representation of an initial molecular mask enlarged to accommodate missing area's in the model.
  • the program MAMA Karlinsky, 1994
  • O Jobones et l , 199]
  • Figure 18 is a representation of an enlargement of the model during the bootstrapping procedure plotted as a function of the expansion step.
  • the number of C" atoms inco ⁇ orated in the model per monomer is given ( — Q — ) as well as the number of correct side chains (— ° ⁇ ). Note that after the first round of building in the molecular replacement map (expansion step 'mr'). 37 residues from the molecular replacement search probes had to be deleted from the model reducing the number of C" atoms to 294. Subsequent cycles allowed for the model to be expanded by small increments.
  • Figure 19 is a representation of a comparison of the C trace from a monomer core model (shown in magenta) and the complete PPCA monomer (shown in yellow).
  • the core model contained only 294 C atoms.
  • the 452 residue PPCA monomer consists of a core domain and a cap domain.
  • the helical subdomain and the maturation subdomain forming the cap domain have been shown in the figure above.
  • Figure 20A-D is a representation of the resolving power of the bootstrapping procedure showing three different stages in map quality The atomic coordinates of the refined model are visualized with the electron density in Figures 20B.
  • Figures 20A and 20B show the initial 2m
  • the electron density is essentially uninte ⁇ retable
  • Fig. 20C shows twofold averaged 2
  • the density for ⁇ -strand M ⁇ 2 (residues 266-271) has become clearly visible Fig.
  • Figure 21 shows a Ramachandran plot calculated for one monomer from a refined model of a pPPCA Both monomers in the asymmetric unit give essentially equivalent plots
  • Figure 22 shows a schematic of a computer system for PPCA or pPPCA structure determination and or rational drug design
  • Figure 23.1-52 lists the atomic coordinates for the active site of a pPPCA dimer having the ammo acid sequence presented as portions of at least one of 50-76, 144-155, 173-197, 226-253, 226-288, 294-310, 327-344, 338- 350, 366-381 and 423-436 of ( Figure 23 1-23.26) 452 ammo acids (designated 1 -452) of monomer 1, as well as corresponding portions of ( Figure 23 26-23 52) 452 amino acids (designated 1001-1452) of monomer 2
  • the present invention provides methods for expressing, purifying and crystallizing a protective protein/cathepsin A (PPCA) or a precursor protective protein/cathepsin A (pPPCA), where the crystals diffract x-rays with sufficiently high resolution to allow determination of the three-dimensional structure of the PPCA or pPPCA, or a portion or subdomain thereof
  • the three-dimensional structure e g ,as provided on computer readable media of the present invention
  • Such ligands can be synthesized or recombinantly produced and are useful as diagnostic agents or drugs for diagnosing, treating, inhibiting or preventing at least one PPCA- or pPPCA-related pathology
  • the determined structure is made using the PPCA or pPPCA ammo acid sequences and/or atomic coordinate/x- ray diffraction data, which are analyzed to provide atomic model output data corresponding to the three-dimensional structure, e g
  • Structure determination methods are also provided by the present invention for rational drug design (RDD) of PPCA or pPPCA ligands
  • RDD rational drug design
  • Such drug design uses computer modeling programs that calculate different molecules expected to interact with the determined active sites, binding sites, or other structural or functional domains or subdomains of a PPCA or a pPPCA
  • These ligands can then be produced and screened for activity in modulating or binding to a PPCA or pPPCA, according to methods and compositions of the present invention
  • the actual PPCA or pPPCA- gand complexes can optionally be crystallized and analyzed using x-ra ⁇ diffraction techniques
  • the diffraction patterns obtained are similarly used to calculate the three-dimensional interaction of the ligand and the PPCA or pPPCA, to confirm that the ligand binds to. or changes the conformation of.
  • doma ⁇ n(s) or subdoma ⁇ n(s) of the PPCA or pPPCA Such screening methods are selected from assays for at least one biological activity of a PPCA or a pPPCA
  • the resulting ligands, provided by methods of the present invention modulate or bind at least one PPCA or pPPCA and are useful for diagnosing, treating or preventing PPCA- or pPPCA- related pathologies in animals, such as humans
  • Ligands of a particular PPCA or pPPCA can similarly modulate other PPCAs or pPPCAs from other sources such as other eukaryotes
  • a PPCA or pPPCA is also provided as a crystallized protein suitable for x-ray diffraction analysis.
  • the x-ray diffraction patterns obtained by the x-ray analysis are of moderate, to moderately high, to high resolution, e.g.. 30-10, 10-3.5 or 1.5-3.5 A. respectively, with the higher resolutions included. These diffraction patterns are suitable and useful for three-dimensional structure determination of a PPCA or a pPPCA. domain or subdomain thereof. The determination of the three-dimensional structure of a PPCA or pPPCA has a broad-based utility.
  • the three-dimensional structure from one or few PPCAs or pPPCAs can be used to identify ligands that have diagnostic or therapeutic value for at least one PPCA- or pPPCA-related pathology that may involve PPCAs or pPPCAs having different amino acid sequences. Determination of Protein Structures
  • the primary structure is obtained by biochemical methods, either by direct determination of the amino acid sequence from the protein, or from the nucleotide sequence of the corresponding gene or cDNA.
  • the quaternary structure of large proteins or aggregates can also be determined by electron microscopy.
  • x-ray crystallography is preferred. See, e.g., Blundell, infra; Oxender, infra; McPherson, infra; Wyckoff, infra.
  • the first prerequisite for solving the three-dimensional structure of a protein by x-ray crystallography is a well- ordered crystal that will diffract x-rays strongly.
  • the crystallographic method directs a beam of x-rays onto a regular, repeating array of many identical molecules so that the x-rays are diffracted from it in a pattern from which the structure of an individual molecule can be retrieved.
  • Well-ordered crystals of globular protein molecules are large, spherical, or ellipsoidal objects with irregular surfaces, and crystals thereof contain large holes or channels that are formed between the individual molecules. These channels, which usually occupy more than half the volume of the crystal, are filled with disordered solvent molecules.
  • the protein molecules are in contact with each other at only a few small regions. This is one reason why structures of proteins determined by x-ray crystallography are generally the same as those for the proteins in solution.
  • crystals are dependent on a number of different parameters, including pH, temperature, protein concentration, the nature of the solvent and precipitant, as well as the presence of added ions or ligands to the protein.
  • Crystallization robots can automate and speed up the work of reproducibly setting up large numbers of crystallization experiments.
  • a pure and homogeneous protein sample is important for successful crystallization. Proteins obtained from cloned genes in efficient expression vectors can be purified quickly to homogeneity in large quantities in a few purification steps.
  • a protein to be crystallized is preferably at least 93-99% pure according to standard criteria of homogeneity. Crystals form when molecules are precipitated very slowly from supersaturated solutions. The most frequently used procedure for making protein crystals is the hanging-drop method, in which a drop of protein solution is brought very gradually to supersaturation by loss of water from the droplet to the larger reservoir that contains salt or polyethylene glycol solution.
  • Different crystal forms can be more or less well-ordered and hence give diffraction patterns of different quality.
  • X-rays are electromagnetic radiation at short wavelengths, emitted when electrons jump from a higher to a lower energy state.
  • x-rays are produced by high-voltage tubes in which a metal plate, the anode, is bombarded with accelerating electrons and thereby caused to emit x-rays of a specific wavelength, so-called monochromatic x-rays.
  • the high voltage rapidly heats up the metal plate, which therefore has to be cooled
  • Efficient cooling is achieved by so-called rotating anode x-ray generators, where the metal plate revolves during the experiment so that different parts are heated up
  • More powerful x-ray beams can be produced in synchrotron storage rings where electrons (or positrons) travel close to the speed of light These particles emit very strong radiation at all wavelengths from short gamma rays to visible light
  • Polychromatic x-ray beams are produced by having a broad window that allows through x-ray radiation with wavelengths of 0 2 - 3 5
  • a ln diffraction experiments a narrow and parallel beam of x-rays is taken out from the x-ray source and directed onto the crystal to produce diffracted beams
  • the incident primary beam causes damage to both protein and solvent molecules
  • the crystal is, therefore, usually cooled to prolong its lifetime (e g , -220 to -50°C)
  • the primary beam must strike the crystal from many different directions to produce all possible diffraction spots, and so the crystal is rotated in the beam during the experiment
  • the diffracted spots are recorded either on a film, the classical method, or by an electronic detector
  • the exposed film has to be measured and digitized by a scanning device, whereas electronic detectors feed the signals they detect directly in a digitized form into a computer
  • Electronic area detectors an electronic film significantly reduce the time required to collect and measure diffraction data
  • the diffraction pattern obtained in an x-ray experiment is related to the crystal that caused the diffraction X- rays that are reflected from adjacent planes travel different distances, and diffraction only occurs when the difference in distance is equal to the wavelength of the x-ray beam This distance is dependent on the reflection angle, which is equal to the angle between the primary beam and the planes
  • each atom in a crystal scatters x-rays in all directions, and only those that positively interfere with one another, according to Bragg's law, give rise to diffracted beams that can be recorded as a distinct diffraction spot above background
  • Each diffraction spot is the result of interference of all x-rays with the same diffraction angle emerging from all atoms
  • each of the about 20.000 diffracted beams that have been measured contain scattered x-rays from each of the around 1500 atoms in the molecule
  • the mathematical tool that is used to handle such problems is called the Fourier transform
  • Each diffracted beam which is recorded as a spot on the film, is defined by three properties the amplitude, which we can measure from the intensity of the spot, the wavelength, which is set by the x-ray source and the phase which is lost in x-ray experiments All three properties are needed for all of the diffracted beams, in order to determine the position of the atoms giving rise to the diffracted beams
  • Phase differences between diffracted spots can be determined from intensity changes following heavy-metal substitution
  • the intensity differences are used to deduce the positions of the heavy atoms in the crystal unit cell
  • Fourier summations of these intensity differences give maps of the vectors between the heavy atoms, the so-called Patterson maps From these vector maps the atomic arrangement of the heavy atoms is deduced From the positions of the heavy metals in the unit cell one can calculate the amplitudes and phases of their contribution to the diffracted beams of protein crystals containing heavy metals
  • the initial model w ill contain some errors Provided the protein crystals diffract to high enough resolution (e g better than 3 5 A) most or substantially all of the errors can be removed by crystal lographic refinement of the model using computer algorithms
  • the model is changed to minimize the difference between the expe ⁇ mentalK observed diffraction amplitudes and those calculated for a hypothetical crystal containing the model (instead of the real molecule)
  • This difference is expressed as an R factor (residual disagreement) which is 0 0 for exact agreement and about 0 59 for total disagreement ln general, the R factor is preferably between 0 15 and 0 35 (such as less than about 024-028) for a well- determined protein structure
  • the residual difference is a consequence of errors and imperfections the data These derive from various sources, including slight variations in the conformation of the protein molecules, as well as inaccurate corrections both for the presence of solvent and for differences in the orientation of the icrocrystals from which the crystal is built This means that the final model represents an average of molecules that
  • Electron-density maps with this resolution range are preferably inte ⁇ reted by fitting the known ammo acid sequences into regions of electron density in which individual atoms are not resolved
  • a PPCA or pPPCA polypeptide can refer to any subset of a PPCA or pPPCA as a domain, subdomain, fragment, consensus sequence or repeating unit thereof
  • a PPCA or pPPCA polypeptide of the present invention can be prepared by, e g (a) recombinant DNA methods,
  • a biological activity of PPCA or pPPCA can be screened according to known screening assays
  • the minimum peptide sequence to have activity is based on the smallest unit containing or comprising a particular domain subdomain, fragment, region, consensus sequence, or repeating unit thereof, having at least one biological activity of a PPCA or pPPCA, such as protecting activity, inhibiting activity or enzyme activity
  • Non-limiting examples of such activities are protecting activity for ⁇ -galactosidase or neuraminidase (NA), modulating activity (inhibition, stimulation or activation) as an for endothelin I (serine carboxypeptidase) or cathepsin A and peptide hydrolyzmg activity (e g substance P and substance P-free acid, oxytocin and oxytocin-free acid,
  • a PPCA or pPPCA includes an association of two or more polypeptide subdomains such as at least one 4 ammo acid portion of a core or cap domain of a PPCA or pPPCA This can include 1 -14 subdomains of the cap domain and/or 1 -44 subdomains of the core domain (as monomers or dimers) or any range value or combination thereof Preferably 1 -4 sets of each of at least one core or cap domains or subdomains are included
  • the structure of a monomer or domain of at least one PPCA includes at least one subdomain of a PPCA of a pPPCA of the present invention can include one or more of the following subdomains, as described herein Generally a PPCA or pPPCA consists of a dimer of a core domain and a cap domain having the following subdomains having the specified residues, e g .
  • a PPCA or pPPCA polypeptide of the invention can have at least 80% homology, such as 80-100% overall homology or identity, with one or more corresponding PPCA or pPPCA subdomains or fragments as described herein, such as a 4-542 ammo acid fragment or portion of the amino acid sequence of Figures 13, 14 or 15
  • the above configurations of subdomains are provided as part of a PPCA or pPPCA polypeptide of the invention, when expressed in a suitable host cell, or otherwise synthesized, to provide at least one structural or functional feature of a native PPCA or pPPCA, such as at least one PPCA-related biological activity
  • Such activities can be assayed using a suitable assay, to establish at least one PPCA biological activity of one or more PPCAs or pPPCAs of the invention
  • a PPCA or pPPCA polypeptide of the invention is not naturally occurring or is naturally occurring but is in a purified or isolated form which does
  • Percent homology or identity can be determined, for example, by comparing sequence information using the
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J Mol Biol 48 443 (1970), as revised by Smith and Waterman (Adv Appl Math 2 482 (1981) Briefly, the GAP program defines similarity as the number of aligned symbols (I e , nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences
  • the preferred default parameters for the GAP program include (1 ) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of G ⁇ bskov and Burgess, Nucl Acids Res 14 6745 (1986), as described by Schwartz and Dayhoff, eds , ATLAS OF PROTEIN
  • Non-limiting examples of substitutions of a PPCA or pPPCA domains or polypeptide of the invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue added in its place according to the following Table 2
  • the types of substitutions which can be made in the protein or peptide molecule of the invention can be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such those presented in Figure 15 Based on such an analysis, alternative substitutions are defined herein as exchanges within one of the following five groups
  • deletions and additions, and substitutions according to the invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule "Characteristics' is defined in a non-inclusive manner to define both changes in secondary structure, e g ⁇ -helix or ⁇ -sheet, as well as changes in physiological activity, e g in biological activity assays
  • PPCA or pPPCA screening assay such as, but not limited to, immunoassays or bioassays, to confirm at least one PPCA or pPPCA biological activity
  • a PPCA and or a pPPCA is now discovered to have serine carboxypeptidase activity and corresponding structural features, although having only about 30% sequence identity to wheat and yeast serine carboxypeptidases
  • carboxypeptidases are members of the hydrolase fold family (Liao et al , Biochemistry 31 9796-9812 (1992), Endnzzi etal, Biochemistry 33 1 1 106-11120 (1994), Ollis et al Protem Eng 5 197-21 1 (1992))
  • the serine carboxypeptidases have peptidase activity at acidic pH ( pH 4 5-5 5) as well as deamidase and esterase activities at pH 7 (reviewed in Breddam et al Carlsberg Res Commun 51 83-128 (1986), Rawlmgs & Barrett, Methods in Enzymology 244 19-61 (1994)) Mutagenesis studies and enzymatic assays have revealed that only the mature form of PPCA possesses a serine carboxypeptide activity
  • a nucleic acid sequence encoding a PPCA or a pPPCA can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases Techniques for such manipulations are disclosed, e g , Sambrook et al , Molecular Cloning A Laboratory Manual, Second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), and Ausubel et al ,Current Protocols tn Molecular Biology, Wiley Interscience, N Y , ( 1988- 1995) and are well known in the art
  • a nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as a PPCA , pPPCA or fragment thereof, in recoverable amounts
  • the precise nature of the regulatory regions needed for gene expression can vary from organism to organism, as is well known in the analogous art See, e g , Sambrook, infra and Ausubel, infra
  • the invention accordingly encompasses the expression of a PPCA or a pPPCA, in either prokaryotic or eukaryotic cells, although eukaryotic expression is preferred
  • Preferred hosts are bacterial or eukaryotic hosts including bacteria yeast, insects, fungi, bird and mammalian cells either in vivo, or tn situ, or host cells of mammalian, insect, bird or yeast origin It is prefe ⁇ ed that the mammalian cell or tissue is of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell can be used
  • Eukaryotic hosts can include yeast, insects, fungi, and mammalian cells either in vivo, or in tissue culture Preferred eukaryotic hosts can also include, but are not limited to insect cells, mammalian cells either in vivo, or in tissue culture Preferred mammalian cells include Xenopus oocytes, HeLa cells, cells of fibroblast origin such as VERO or CHO- 1 or cells of lymphoid origin and their derivatives
  • Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites
  • Mammalian cells which can be useful as hosts include cells of fibroblast origin such as but not limited to NIH 3T3 VERO or CHO or cells of lymphoid origin, such as but not limited to the hybridoma SP2/0-Agl4 or the murine myeloma P3-X63Ag8 hamster cell lines (e g , CHO-K1 and progenitors, e CHO- DUXB 1 1 ) and their derivatives
  • One preferred type of mammalian cells are cells which are intended to replace the function of the genetically deficient cells in vivo Neuronally derived cells are preferred for gene therapy of disorders of the nervous system
  • a mammalian cell host many possible vector systems are available for the expression of at least one PPCA or pPPCA
  • a wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the host The transcription
  • baculoviral vectors are presently preferred hosts for large scale PPCA or pPPCA production according to the invention
  • Production of PPCA or pPPCA in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express transmembrane polypeptide by methods known to those skilled in the related arts See Ausubel infra ⁇ 16 8-16.1 1
  • the introduced nucleotide sequence will be inco ⁇ orated into a plasmid or viral vector capable of autonomous replication in the recipient host
  • a plasmid or viral vector capable of autonomous replication in the recipient host
  • Any of a wide variety of vectors can be employed for this pu ⁇ ose See, e g , Ausubel et a! , infra, ⁇ 1 5, 1 10, 7 1 , 7 3, 8 1, 9 6, 9 7, 13 4, 16 2.
  • Factors of importance in selecting a particular plasmid or viral vector include the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells which do not contain the vector, the number of copies of the vector which are desired in a particular host and whether it is desirable to be able to "shuttle" the vector between host cells of different species
  • Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e , glycosylation, cleavage) of proteins
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed
  • expression in a bacterial system can be used to produce an unglycosylated core protein product Expression in yeast will produce a glycosylated product Expression in mammalian cells can be used to ensure "native" glycosylation of the heterologous PPCA or pPPCA
  • different vector/host expression systems can effect processing reactions such as proteolytic cleavages to different extents
  • the DNA construct(s) can be introduced into an appropriate host cell by any of a variety of suitable means, I e , transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells
  • Expression of the cloned gene molecule(s) results in the production of a PPCA or pPPCA This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example by administration of bro
  • a PPCA or pPPCA. or fragments thereof of this invention can be obtained by expression from recombinant DNA according to known methods Alternatively a PPCA or pPPCA can be purified from biological material A PPCA or a pPPCA can be purified from different mammalian tissues (e g human placenta rat liver mouse liver, pig k ⁇ dne>, bovine testes, bovine liver, and the like) of various genus and species
  • the PPCA or pPPCA can be isolated and purified in accordance with conventional method steps such as extraction precipitation, chromatography affinity chromatography electrophoresis, or the like
  • cells expressing at least one PPCA or pPPCA in suitable levels can be collected by centrifugation or with suitable buffers
  • a pPPCA or PPCA can be isolated by the use of antibodies, such as, but not limited to, a PPCA- or pPPCA-specific antibody Such antibodies can be obtained by known method steps (see, e g Harlow and Lane ANTIBODIES A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988).
  • a PPCA or a pPPCA can be purified from different mammalian tissues (e g , human placenta, rat liver, mouse liver, pig kidney, bovine testes, bovine liver, and the like) of various genus and species, using known techniques such as gel filtration, phase separation and affinity chromatography, e g .using polyclonal or monoclonal antibodies specific for a PPCA or pPPCA, according to known methods See , e g Oxender et al Protein Engineering, Liss, New York (1986)
  • a PPCA or pPPCA is isolated in soluble form in sufficient purity and concentration (e g , a monomer or dimer) for crystallization
  • the PPCA or pPPCA is then isolated and assayed for biological activity (e g , cathepsin A) and for lack of aggregation (which interferes with crystallization)
  • the purified PPCA or pPPCA preferably runs as a single band for each monomer under reducing or nonreducing polyacrylamide gel electrophoresis (PAGE) (nonreducing is used to evaluate the presence of cysteine bridges)
  • the purified PPCA or pPPCA is preferably crystallized under varying conditions of at least one of the following pH, buffer type, buffer concentration, salt type, polymer type, polymer concentration, other precipitating ligands and concentration of purified PPCA or pPPCA
  • pH, buffer type, buffer concentration, salt type, polymer type, polymer concentration, other precipitating ligands and concentration of purified PPCA or pPPCA See, e g , known methods (Blundell et al , Protein Crystallography, Academic Press, London (1976), Oxender, infra, McPherson, The Preparation and Analysis of Protein Crystals, Wiley lnterscience, N Y (1982)) or methods provided in a commercial kit, such as CRYSTAL SCREEN (Hampton Research, Riverside, CA)
  • the crystallized PPCA protein can optionally be tested for at least one PPCA activity and differently sized and shaped crystals are further tested for suitability for x-ray dif
  • the hanging drop method is preferably used to crystallize the purified protein See, e g , Blundell, infra, Oxender, infra, McPherson, infra. Wyckoff, infra, Taylor et al . J Mol Biol 226 1287-1290 (1992), Takimoto et al ( 1992), infra, CRYSTAL SCREEN, Hampton Research
  • a mixture of the purified protein and precipitant can include the following
  • buffer type e , tromethamine (TRIZMA), sodium azide (NaN 3 ), phosphate, sodium, or cacodylate acetates, imidazole, Tris HCI, sodium hepes
  • buffer concentration e g , 1-100 M
  • salt type e g , sodium azide, calcium chloride, sodium citrate, magnesium chloride, ammonium acetate, ammonium sulfate, potassium phosphate, magnesium acetate, zinc acetate, calcium acetate
  • polymer type and concentration e g polyethylene glycol (PEG) 1 -50%, type 400- 10,000).
  • the above mixtures are used and screened by varying at least one of pH, buffer type, buffer concentration, precipitating salt type or additive or their concentrations, PEG type, PEG concentration, and protein concentration Crystals ranging in size from 0 1-0 9 mm are formed in 1 -14 days These crystals diffract x-rays to at least 10 A resolution, such as 0 15-10 0 A, or any range of value therein, such as 1 5, 1 6, 1 7 1 8, 1 9, 2 0, 2 1, 2 2, 2 3, 2 4, 2 5, 2 6, 2 7, 2 8, 2 9, 3 0, 3 1 , 3 2, 3 3, 3 4 or 3 5, with 3 5 A or higher being preferred for the highest resolution In addition to diffraction patterns having this highest resolution, lower resolution, such as 25-3 5 A can also be used See e g , Blundell, infra, Oxender, infra.
  • Other washed crystals are preferably run on a gel and stained, and those that migrate in the same position as the purified PPCA or pPPCA are preferably used From two to one hundred crystals are observed in one drop and crystal forms can occur, such as, but not limited to, orthorombic, bipyramidal, rhomboid, and cubic
  • Initial x-ray analyses indicate that such crystals diffract at moderately high to high resolution When fewer crystals are produced in a drop, they can be much larger size, e g , 0 4-0 9 mm See, e g , Blundell, infra. Oxender, infra, McPherson, infr
  • Crystals so produced for a PPCA or pPPCA are x-ray analyzed using a suitable x-ray source Diffraction patterns are obtained Crystals are preferably stable for at least 10 hrs in the x-ray beam Frozen crystals (e g , -220 to -50°C) are optionally used for longer x-ray exposures (e , 5-72 hrs), the crystals being relatively more stable to the x-rays in the frozen state To collect the maximum number of useful reflections, multiple frames are optionally collected as the crystal is rotated in the x-ray beam, e g , for 5-72 hrs Larger crystals (>02 mm) are preferred, to increase the resolution of the x-ray diffraction patterns obtained Crystals are preferably analyzed using a synchrotron high energy x-ray source Using frozen crystals, x-ray diffraction data is collected on crystals that diffract to at least a relatively high resolution of 10-1 5 A, with lower resolutions also useful, such as 25-
  • Passing an x-ray beam through a crystal produces a diffraction pattern as a result of the x-rays interacting and being scattered by the contents of the crystal
  • the diffraction pattern can be visualized using, e g an image plate or film, resulting in an image with spots corresponding to the diffracted x-rays
  • the positions of the spots in the diffraction pattern are used to determine parameters intrinsic to the crystal (such as unicell parameters) and to gam information on the packing of the molecules in the crystal
  • the intensity of the spots contains the Fourier transformation of the molecules in the crystal. / e information on each atom in the crystal and hence of the crystallized molecule
  • the data is processed This includes measuring the spots on each diffraction pattern in terms of position and intensity
  • This information is processed (i e mathematical operations are performed on the data (such as scaling merging and converting the data from intensity of diffracted beams to amplitudes)) to yield a set of data which is in a form as can be used for the further structure determination of the molecule crystallized
  • the amplitudes of the diffracted x-ravs are then combined with calculated phases to produce an electron density map of the contents of the crystal In this electron density map the structure of the molecules (as present in the crystal) is built
  • the phases can be determined with various known techniques, one being molecular replacement
  • the phases can be further optimized using a technique called density modification, which allows electron density maps of better quality to be produced facilitating inte ⁇ retation and model building therein
  • density modification allows electron density maps of better quality to be produced facilitating inte ⁇ retation and model building therein
  • the atomic model is then refined by allowing the atoms in the model to move in order to match the diffraction data as well as possible while continuing to satisfy stereochemical constraints (sensible bond lengths, bond angles and the like) See, e g , Blundell, infra, Oxender, infra, McPherson, infra, Wyckoff, infra, Computer Related Embodiments
  • An amino acid sequence of a PPCA or pPPCA and/or atomic coordinate/x-ray diffraction data, useful for computer structure determination of a PPCA, pPPCA or a portion thereof, can be "provided” in a variety of mediums to facilitate use thereof
  • provided refers to a manufacture, which contains a PPCA or pPPCA ammo acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention, e g , the amino sequence provided in Figures 13-15, a representative fragment thereof, or an ammo acid sequence having at least 80-100% overall identity to a 5-542 ammo acid fragment of an am o acid sequence of Figures 13-15
  • Such a method provides the ammo acid sequence and/or atomic coordinate/x-ray diffraction data in a form which allows a skilled artisan to analyze and determine the three- dimensional structure of a PPCA, a pPPCA or a subdomain thereof
  • PPCA, pPPCA, or at least one subdomain thereof, amino acid sequence and/or atomic coord inate/x-ray diffraction data of the present invention is recorded on computer readable media
  • computer readable media refers to any medium which can be read and accessed directly by a computer Such media include, but are not limited to magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape, optical storage media such as optical discs or CD-ROM, electrical storage media such as RAM and ROM, and hybrids of these categories such as magnetic/optical storage media
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as optical discs or CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media
  • any of the presently known computer readable media can be used to create a manufacture comprising computer readable medium having recorded thereon an amino acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon an ammo acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention
  • the choice of the data storage structure will generally be based on the means chosen to access the stored information
  • a variety of data processor programs and formats can be used to store the sequence and x-ray data information of the present invention on computer readable medium
  • the sequence information can be represented in a word processing text file formatted in commercially-available software such as WordPerfect and MICROSOFT Word or represented in the form of an ASCII file, stored in a database application, such as DB2, Svbase Oracle or the like
  • a skilled artisan can readily adapt any number of dataprocessor structuring formats (e g text file or database) in order to obtain computer readable medium having recorded thereon the information of the present invention
  • the present invention further provides systems, particularly computer-based systems, which contain the sequence and or diffraction data described herein
  • Such systems are designed to do structure determination and RDD for a PPCA, pPPCA or at least one subdomain thereof
  • Non-limiting examples are microcomputer workstations available from Silicon Graphics Inco ⁇ orated and Sun Microsystems running Unix based Windows NT or IBM OS/2 operating systems
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the sequence and/or atomic coord inate/x-ray diffraction data of the present invention
  • the minimum hardware means of the computer-based systems of the present invention comprises a central processing unit
  • the computer-based systems of the present invention comprise a data storage means having stored therein a PPCA, pPPCA or fragment sequence and/or atomic coordinate/x-ray diffraction data of the present invention and the necessary hardware means and software means for supporting and implementing an analysis means
  • data storage means refers to memory which can store sequence or atomic coordinate/x-ray diffraction data of the present invention, or a memory access means which can access manufactures having recorded thereon the sequence or x-ray data of the present invention
  • search means or “analysis means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence or x-ray data stored within the data storage means Search means are used to identify fragments or regions of a PPCA or pPPCA which match a particular target sequence or target motif
  • search means are used to identify fragments or regions of a PPCA or pPPCA which match a particular target sequence or target motif
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration or electron density map which is formed upon the folding of the target motif
  • Protein target motifs include, but are not limited to, enzymic active sites, structural subdomains, epitopes, functional domains and signal sequences
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention
  • a variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify structural motifs or mte ⁇ ret electron density maps derived in part from the atomic coord ⁇ nate/x-ra> diffraction data
  • any one of the publicly available computer modeling programs can be used as the search means for the computer-based systems of the present invention
  • Figure 22 provides a block diagram of a computer system 102 that can be used to implement the present invention
  • the computer system 102 includes a processor 106 connected to a bus 104
  • main memory 108 preferably implemented as random access memory. RAM
  • secondary storage memory 1 such as a hard drive 1 12.
  • a removable storage medium 1 such as a hard drive 1 12.
  • the removable medium storage device 1 14 may represent, for example a floppy disk drive, a CD-ROM drive a magnetic tape drive, etc
  • a removable storage medium 1 16 (such as a disk a compact disk a magnetic tape etc ) containing control logic and/or data recorded therein may be inserted into the removable medium storage medium 1 14
  • the computer system 102 includes appropriate software for reading the control logic and/or the data from the removable medium storage device 1 14 once inserted in the removable medium storage device 114
  • Amino acid, encoding nucleotide or other sequence and/or atomic coordmate/x-ray diffraction data of the present invention may be stored in a well known manner in the main memory 108, any of the secondary storage devices 1 10, and/or a removable storage device 1 16
  • Software for accessing and processing the amino acid sequence and/or atomic coordinate/x-ray diffraction data reside in main memory 108 during execution
  • the monitor 120 is optionally used to visualize the structure data Structure Determination
  • One or more computational steps, computer programs and/or computer algorithms are used to build a molecular
  • 3-D model of a PPCA or pPPCA using amino acid sequence data from Figures 13-15 (or variants thereof) and/or atomic coordinate/x-ray diffraction data, as presented herein ln x-ray crystallography, x-ray diffraction data and phases are combined to produce electron density maps in which the three-dimensional structure of a PPCA or pPPCA is then built or modeled This structure can then be used for RDD of modulators of at least one PPCA- or pPPCA-related activity that is relevant to at least one PPCA- or pPPCA-related pathology
  • Electron density maps can be calculated using such programs as those from the CCP4 computing package (SERC (UK) Collaborative Computing Project 4, Daresbury Laboratory, UK, 1979) Cycles of two-fold averaging can further be used, such as with the program RAVE (Kleywegt & Jones, Bailey et al eds , First Map to Final Model, SERC Daresbury Laboratory, UK, pp 59-66 (1994)) and gradual model expansion
  • RAVE Ket & Jones, Bailey et al eds , First Map to Final Model, SERC Daresbury Laboratory, UK, pp 59-66 (1994)
  • Rigid body and positional refinement can be carried out using a program such as X-PLOR (Br ⁇ nger (1992), infra), e g , with the stereochemical parameters of Engh and Huber (Acta Cryst A47 392-400 (1991)) If the model at this stage in the averaged maps still misses residues (e g , at least 5-10 per subunit), the some or all of the missing residues can be inco ⁇ orated in the model during additional cycles of positional refinement and model building
  • the refinement procedure can start using data from lower resolution (e g , 25-l ⁇ A to 10-3 0 A and then gradually extended to include data from 12- ⁇ A to 3 0-1 5 A B-values (also termed temperature factors) for individual atoms can be refined once data of 2 ⁇ A or higher (e g up to 1 5 A) has been added Subsequently waters can be gradually added
  • a program such as ARP (La zin and Wilson, Acta Cryst D
  • the determination of the three-dimensional structure of a PPCA or pPPCA, as described herein, provides a basis for the design of new and specific ligands for the diagnosis and/or treatment of at least one PPCA- or pPPCA- related pathology
  • Several approaches can be taken for the use of the crystal structure of a PPCA or pPPCA in the rational design of ligands of this protein
  • a computer-assisted, manual examination of the active site structure is optionally done
  • the use of software such as GRID ( Goodford, J Med Chem 28 849-857 (1985)) a program that determines probable interaction sites between probes with various functional group characteristics and the enzyme surface — is used to analyze the active site to determine structures of inhibiting compounds
  • the program calculations with suitable inhibiting groups on molecules (e g , protonated primary amines) as the probe, are used to identify potential hotspots around accessible positions at suitable energy contour levels Suitable ligands, as inhibiting or stimulating modulating compounds or compositions are then
  • a diagnostic or therapeutic PPCA or pPPCA modulating ligand of the present invention can be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof or any combination thereof, which can be detectably labeled as for labeling antibodies
  • Such labels include, but are not limited to.
  • any other known diagnostic or therapeutic agent can be used in a method of the invention After preliminary experiments are done to determine the K m of the substrate with each enzvme activity of a
  • a PPCA or pPPCA ligand is any molecule, compound or composition that is capable of associating with a PPCA or pPPCA and optionally modulating at least one function or structural feature of a PPCA or pPPCA
  • a PPCA or pPPCA ligand modulates at least one biological activity of a PPCA or pPPCA Demonstration of clinically useful levels, e g , in vivo activity is also important
  • PPCA or pPPCA inhibitors for biological activity in animal models e g , rat, mouse, rabbit
  • animal models e g , rat, mouse, rabbit
  • the present invention also provides methods for identifying diagnostic or therapeutic ligands of PPCA or pPPCA via computer RDD, to treat a PPCA-related pathology
  • a method for determining the therapeutic or diagnostic use of a PPCA or pPPCA modulating ligand, to treat a PPCA related pathology comprises the steps of administering a known dose of at least one ligand containing compositions to an animal model having a phenotype corresponding to a PPCA-related pathology, monitoring the appropriate biological or biochemical parameters, and comparing the results with treated animals to those of untreated animals.
  • Results indicating the onset or presence of a PPCA related pathology are generally referred to herein as "symptoms" of the disease See , e g , U S Appl No 08/397,693, filed March 2 1995, which is entirely i ⁇ co ⁇ orated herein by reference
  • Appropriate biological and biochemical parameters that reflect the onset and progression of a PPCA related pathology include but are not limited to, (1 ) gross biological parameters, e g , physical appearance (i e , flattening of the face rough haircoat and/or subcutaneous swelling in affected animals) or growth (reduced weight gam), (2) gross behavioral parameters, e g , lack of coordination, (3) biochemical assays, e g .
  • a first method of evaluating the therapeutic potential of a composition using the transgenic non-human animals of the invention comprises the steps of
  • a second method of evaluating the therapeutic potential of a composition using the non-human animals of the invention comprises the steps of
  • the composition being tested may comprise a chemical compound administered by circulatory injection or oral ingestion
  • the composition being evaluated may alternatively comprise a polypeptide administered by circulatory injection of an isolated or recombinant bacterium or virus that is live or attenuated, wherein the polypeptide is present on the surface of the bacterium or virus prior to injection, or a polypeptide administered by circulatory injection of an isolated or recombinant bacterium or virus capable of reproduction within a non-human animal, and the polypeptide is produced within a non-human animal by genetic expression of a DNA sequence encoding the polypeptide
  • the composition being evaluated may comprise one or more nucleic acids, including a gene from the human genome or a processed RNA transcript thereof
  • the composition being evaluated may comprise cells removed from a mammal and genetically engineered to overexpress a lysosomal protein or some other therapeutic polypeptide
  • the PPCA modulating ligand Once the PPCA modulating ligand has been shown to be effective in an animal model, it can then be tested in human clinical trials, according to known method steps ln the above methods, delivery of the composition being tested to non-human animals is achieved via means appropriate for the composition being tested, e.g., by diet, by intermittent or continuous intravenous injection of one or more of the compositions or of a posome (Rahman and Schem, in Liposomes as Drug Carriers, Grego ⁇ adis, ed , John Wiley, New York ( 1988), pages 381-400, Gabizon, A , in Drug Carrier Systems, Vol 9, Roerdink et al , eds , John Wiley, New York (1989), pages 185-212) or microparticle (Tice et al , U S Patent 4,542,025 (Sep 17, 1985)) formulation comprising one or more of the compositions, via subdermal implantation of drug-polymer conjugates (Duncan
  • the present invention further provides a method for modulating the activity of a PPCA or pPPCA protein in a cell
  • ligands antagonists or agonists
  • ligands which have been identified to inhibit or enhance the activity of at least one PPCA or pPPCA ligand can be formulated so that the ligand can be contacted with a cell expressing at least one PPCA or pPPCA protein in vivo
  • the contacting of such a cell with such a ligand results in the in vivo modulation of at least one biological activity of a PPCA or pPPCA
  • At least one PPCA or pPPCA modulating compound or composition of the invention can be administered by any means that achieve the intended pu ⁇ ose, using a suitable pharmaceutical composition or formulation
  • administration can be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes
  • parenteral administration can be by the oral route
  • Parenteral administration can be by bolus injection or by gradual perfusion over time
  • a typical regimen for treatment or prophylaxis comprises administration of an effective amount over a period of one or several days, up to and including between one week and about six months
  • dosage of a diagnostic/pharmaceutical compound or composition of the invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the diagnostic/ pharmaceutical effect desired
  • the ranges of effective doses provided herein are not intended to be limiting and represent preferred dose ranges However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one skilled in the relevant arts See, e , Berkow et al , eds , The Merck Manual, 16th edition, Merck and Co , Rahway, N J , 1992.
  • the total dose required for each treatment can be administered by multiple doses or in a single dose
  • the diagnostic/pharmaceutical compound or composition can be administered alone or in conjunction with other diagnostics and/or pharmaceuticals directed to the pathology, or directed to other symptoms of the pathology
  • Effective amounts of a diagnostic/pharmaceutical compound or composition of the invention are from about 0 1 ⁇ g to about 100 mg kg body weight, administered at intervals of 4-72 hours, for a period of 2 hours to 1 year, and/or any range or value therein
  • the recipients of administration of compounds and/or compositions of the invention can be any mammals
  • the preferred recipients are mammals of the Orders P ⁇ mata (including humans, apes and monkeys),
  • Arte ⁇ odactyla including horses, goats, cows, sheep, pigs
  • Rodenta including mice, rats, rabbits, and hamsters
  • Camivora including cats, and dogs
  • the present invention provides in one aspect, the determination of the three-dimensional structure of the human protective protein/cathepsin A (PPCA) in the precursor form (pPPCA) by a combination of molecular replacement and twofold density averaging
  • the structure presented here is the first of an enzvme associated with a human PPCA related pathology, and the third human Ivsosomal enzyme structure determined
  • the structure gives us insight into the zymogen activation mechanism of pPPCA . as well as the expected 3-D structure of PPCA and its specific and new enzymatic activities PPCA and p PPCA Expression and Purification
  • Recombinant polyhednn-negative recombinant baculoviruses were then selected and purified by sequential plaque assays, and verified by dot blot and southern blot analysis (Summers et al , 1987) Large quantities of inoculum were produced by infection of insect cells at 25-50 % confluency, with recombinant virus at a multiplicity of infection (MOI) of ⁇ 1 pfu/cell After 3 to 6 days at 27°C, when all cells appeared infected, the medium was harvested and centrifuged for 5 m at 1000 ⁇ m to remove detached cells The titre of the inoculum was determined by plaque assay analysis
  • Sf21 cells were cultured in either 175 CM 2 or 500 CM 2 flasks (triple flask, Nunc) to near confluency, and infected with recombinant baculoviruses at a MOI of 5- 10 pfu/cell After 1 5 h incubation at 27 °C, the inoculum was replaced with complete medium for additional 8 to 10 hrs Cell monolayers were then rinsed with PBS and cultured further for 38 h in unsupplemented Grace's medium After infection the medium was collected, centrifuged for 5 m at 1500 g, and for 1 h at 100 000 g (Beckmann SW-28 rotor) to remove virus particles
  • the antibodies designated anti-pep, were tested on immunoblots and by immunoprecipitations of baculovirus produced PPCA
  • Blots were incubated for at least 12 h in blocking buffer (0 01 M tns-buffered saline pH 8 0 (TBS) 0 05%
  • Crystallization of PPCA Fractions containing the precursor form of the protein as assayed on an SDS-PAGE gel were pooled Subsequently the protein was concentrated to 5 mg/ml and the buffer exchanged to 50 mM NaAc pH 5 2 or 50 mM MES pH 6 5 using a CENTRICON- I 0 Crystals were grown using the hanging drop vapor diffusion technique Crystals suitable for data collection were grown using a reservoir solution containing 2- 10 % PEG 8000, pH 8 0 - 8 3, 50mM TRIZMA, ImM NaN 3 , 025 % ⁇ -octyl glucoside at 4-12°C Mixing non-equal volumes of protein solution (in the range 5-10 ⁇ l) and reservoir solution ( in the range 2-6 W) enhanced the occurrence of single large crystals per drop under these crystallization conditions The concentration of the protein solution before mixing was 5 mg/ml Crystal growth was enhanced by macrocrystallization techniques (anything that promotes growth of big crystals) and in some
  • Example 2 Structure Determination ofapPPCA Crystallized from Human Cells Data Collection, Data Processing and Reduction To allow for data collection at cryotemperatures, the crystals were cryoprotected by adding glycerol in 5% -10% steps to a solution of about 12% PEG 8000, 50 mM TRIZMA, pH 8 0, ImM NaN 3 , 0 25% ⁇ -octyl glucoside, which served as an artificial mother liquor The crystals were incubated for half an hour at 40"C after each addition of glycerol The final mother liquor contained 30% glycerol Gradually increasing the glycerol was needed to help keep the crystals from cracking
  • the 'multi-Ala core' search model was constructed from the atomic coordinates of the CPW monomer (Liao et al , 1992), based on the sequence alignment as presented in Figure 15 Regions expected to deviate in structure between PPCA and CPW were deleted from the model (1 e with low sequence identity or located in loops) The 125 residues identical in PPCA and CPW were left in the model, 1 12 residues were truncated to alanine The remaining 94 residues through differing between CPW and PPCA, were considered sufficiently similar in size and the CPW residue left as such in the model The resulting 'multi-Ala core' monomer consisted of 331 residues, constituting a large portion of the core domain and little atomic information for the 'cap' domain (see Figure 1 ) The model contained 30% of the expected protein scattering mass given the fact that there are two monomers in the asymmetric unit The sequence identity between this search model and the true PPCA structure was 37 7% Rotation Function, PC
  • SigmaA weighted map which we called the 'best monomer map' Averaging: Search for Missing Density : The phasing power from the rigid body refined 'best monomer cores' consisting of 294 residues per core was insufficient to bring back inte ⁇ retable electron density for the missing part of the model.
  • Model Building A conservative model building strategy was adopted Initially only side chains were mutated in the core region to fit the PPCA ammo acid sequence and where the density was clear, poly-alanine fragments were built in the insertion area's (loops and the cap domain) Newly included atoms were given a B-factor of 20 A 2 Only once models bmc5 and bmc6 were obtained, was the electron density of sufficient quality to allow side chains to be inco ⁇ orated confidently in the cap domain (residues 1 0 - 303) At this stage the C trace was virtually complete for the whole dimer and the sequence could be fit unambiguously
  • Positional refinement was postponed until after 3 cycles of bootstrapping resulting in a final model containing 91% of the C atoms Forty steps of positional refinement were then carried out to improve the geometry of the model Subsequently only one of the refined monomer was taken and the other generated using NCS operators The rational for delaying the positional refinement is addressed in the discussion
  • the program ARP was used to check our model, in particular the region at the dimer interface (Lamzin & Wilson, 1993) Prior to the final round of positional refinement, an IF ob! I/ ⁇ cutoff was applied to reject 10% of the weakest data as well as an anisotropic scale factor to offset the decreased resolution along the crystallographic a axis
  • the final model is of good geometry with a final R f ⁇ or of 21 3% (R fr ⁇ of 26 8 %) for data between 8 0 and 2 2 A (see Table 3)
  • a Ramachandran plot is given in Figure 21
  • the r m s coordinate error is 0 282 as calculated by SigmaA (Read, 1986)
  • the average phase difference between the initial molecular replacement model and the currently refined model is calculated to be 71° for data between 10 - 2 2 A
  • the structure determination of PPCA is special in that two-fold averaging could be applied to refine very poor molecular replacement phases, enabling us to retrieve electron density for 148 residues and 185 side chains per monomer ln total 314 complete residues were added per asymmetric unit, equivalent to about 35 kDa of protein
  • a number of factors contributed to a successful structure determination Crystal Packing Each monomer in the crystal is interacting with four non-crystallographically related monomers By far the most extensive contact is with a non-crystallographically related monomer generating the physiological dimer
  • Three additional contacts are extensive crystal contacts ranging from 200-800 A 2 averaged per monomer
  • the largest nondimer crystal contact involves the precursor loops from two crystallographically independent monomers ( region 265-267, 281-295 from monomer 1 with residues 281 -293 from monomer 2) making intimate contact with each other Summed together these loops create an intermolecular buried surface of 1680 A 2 We believe that this stabilizes an otherwise very flexible area, possibly explaining the good
  • the electron density in this region is of very good quality with average temperature factors of 16 6 A : for main chain and 18 3 A 2 for side chains pPPCA and the Hydrolase Family
  • the fold of pPPCA belongs to the large hydrolase fold family containing enzymes such as the serine carboxypeptidases, dehalogenase, various lipases and acetylcholine esterase (Ol s et al ( 1992), infra), having various different catalytic functions
  • the central core is the same (a central ⁇ -sheet flanked by ⁇ -helices on both sides) the proteins in this family all seem to have different 'cap' domains, both with respect to fold as well as size ( Figure 7A-F)
  • pPPCA has one of the largest cap domains comprising 121 residues forming the three helical bundle of the helical subdomain and a three stranded ⁇ -sheet of the maturation subdomain
  • the overall fold of the pPPCA monomer is similar to that of the wheat and yeast serine carboxypeptidases (End ⁇ zzi et al (1994), infra, OIlis et al (1992), infra)
  • the complete core domains of pPPCA and CPW superimpose with an r m s deviation of I 7 A for 302 C ⁇ atoms and 38% sequence identity
  • Deleting major deviating loops from the core domain allows for pPPCA to superimpose with an r m s deviation of 1 2 A onto CPW and CPY (293 equivalent C's with 40 % sequence identity for CPW/pPPCA and 271 equivalent C's for CPY/pPPCA with 42 2% identity)
  • the cap domain in pPPCA differs significantly from the CPW and CPY counte ⁇ arts
  • the pPPCA structure reveals a large maturation subdomain not present in the structure of CPW and CPY for which the structures of the enzvmatically active forms are known All three enzymes contain a 3 helical bundle in the cap domain The sequence identity between the three proteins in this region is very low (ca 12 %)
  • PPCA shows a much greater deviation Hal superimposes reasonably well with the CPW counte ⁇ art maintaining the same general orientation with respect to the core domain (requiring a rotation of only 74°)
  • the oxyanion hole proposed to stabilize the negatively charged tetrahedral intermediate in serine carboxypeptidases is formed by the backbone amides of Gly 57 and Tyr 151 in PPCA
  • the 32 atoms of the catalytic triad residues plus the oxyanion hole amides from PPCA, CPY and CPW superimpose with an r.m s deviation of 04 A indicating the very high degree of structural similarity of the active site in the PPCA precursor with those in the fully active enzymes CPY and CPW, (see Table 4)
  • the carboxylate of Asp 372 and the i idazole of His 429 in PPCA are non-planar, making an angle of approximately 60° between the imidazole and the carboxylate A similar non-plana ⁇ ty has been observed in CPW and CPY, in contrast to the planar orientation found in subtilisin- and trypsm-rype serine proteases (McPhalen et al Bio
  • Arrival in the endosome/lysosome is expected to result in protonation of either the Asp or the Glu residue or both, resulting in unfavorable electrostatic interactions and destabilization of this charge cluster. This in turn is expected to promote partial unfolding of maturation subdomain, allowing easier access to additional potential cleavage sites, and stimulating removal of the 'blocking' peptide which fills the active site in the precursor.
  • the maturation mechanism for pPPCA appears to be novel among proteases for which the three-dimensional structure of the zymogen is known.
  • the catalytic triad in the precursor form is in a catalytically competent conformation. Enzymatic activity is prevented by a 'blocking " peptide.
  • the blocking peptide is however different from the excision peptide and does not get excised from the mature enzyme. This leads to the distinct difference with the other known maturation mechanisms in that, after disappearance of the excision peptide, up to 35 residues filling the active site cleft in the PPCA precursor must rearrange to render the catalytic triad solvent accessible (see Figure 12), but do not get cleaved off.
  • the catalytic triad is housed in the core domain and the various cap domains attenuate the biological function by influencing entirely different properties such as: (I) enzyme kinetics exemplified by the interfacial activation of lipases (Smith et al, Curr. Opinion in Structural Biology 2:490-496 ( 1992)); (ii) substrate channeling as is proposed for acetylcholine esterase (Sussman et al. (1991 ), infra); (iii) substrate recognition, proposed for dehalogenase by (Franken et al.
  • PPCA protective protein/cathepsin A
  • PPCA has a 30% sequence identity to the wheat serine carboxypeptidase (CPW) and yeast serine carboxypeptidase (CPY). It has been show that PPCA in the precursor form is inactive, but upon maturation, entailing excision of a 2 kDa peptide, carboxypeptidase activity is released.
  • CPW wheat serine carboxypeptidase
  • CPY yeast serine carboxypeptidase
  • the precursor structure reveals an inactivation mechanism that has not been seen before in any of the other known zymogen structures of proteases (available for the serine-. metallo- and aspartic protease classes).
  • the catalytic triad seems to have an arrangement poised for catalysis. However, the triad is rendered solvent and substrate inaccessible by a strand from the maturation subdomain binding in the active site cleft. Su ⁇ risingly, this strand called the 'blocking' peptide does not overlap with the 2 kDa excision' peptide.

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Abstract

On décrit la protéine/cathepsine A (PPCA) protectrice et cristalllisée, un de ses précurseurs (pPPCA) et au moins un de ses sous-domaines, des procédés d'analyse par diffraction des rayons X qui permettent de produire des motifs de diffraction des rayons X à résolution assez élevée pour la détermination de la structure tridimensionnelle de la protéine, ainsi que des procédés d'élaboration rationnelle de substances thérapeutiques, basés sur l'utilisation de données de séquences d'acides aminés et/ou de données de cristallographie aux rayons X fournies sur des supports lisibles par ordinateur puis analysées sur un système informatique doté d'algorithmes de calcul appropriés.
PCT/US1996/017325 1995-10-26 1996-10-25 Proteine/cathepsine a protectrice et precurseur: cristallisation, diffraction des rayons x, determination de structure tridimensionnelle et elaboration rationnelle de substances therapeutiques WO1997015588A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009148A1 (fr) * 1997-08-13 1999-02-25 Vertex Pharmaceuticals Incorporated Cristaux de l'helicase du virus de l'hepatite c ou des fragments de celle-ci comprenant une poche de fixation de l'helicase
WO1999019465A1 (fr) * 1997-10-14 1999-04-22 E.I. Du Pont De Nemours And Company Trihydroxynaphtalene reductase: procedes permettant la determination de sa structure tridimensionnelle et la mise au point rationnelle de ses inhibiteurs
WO1999040182A3 (fr) * 1998-02-04 1999-10-07 Immunex Corp Enzyme de conversion cristalline du facteur tnf alpha et ses utilisations
WO2001055443A1 (fr) * 2000-01-31 2001-08-02 Pharmacia & Upjohn Company Cristallisation et determination de la structure de staphylococcus aureus nad synthetase
WO2002020804A1 (fr) * 2000-09-08 2002-03-14 Prozymex A/S Structure de cristal peptidase dipeptidyl i et son utilisation
WO2002075277A3 (fr) * 2001-03-16 2003-02-20 Irm Llc Procede et appareil permettant d'effectuer des etapes de traitement multiples sur un echantillon dans une cuve unique
US6842704B2 (en) 1998-02-04 2005-01-11 Immunex Corporation Crystalline TNF-α-converting enzyme and uses thereof
WO2005047898A3 (fr) * 2003-10-24 2005-09-15 Gilead Sciences Inc Procedes et compositions pour l'identification des composes therapeutiques
US7383135B1 (en) 1998-05-04 2008-06-03 Vertex Pharmaceuticals Incorporated Methods of designing inhibitors for JNK kinases
US7736875B2 (en) 2000-09-08 2010-06-15 Prozymex A/S Dipeptidyl peptidase I crystal structure and its uses
US9399791B2 (en) 2011-08-31 2016-07-26 St. Jude Children's Research Hospital Methods of treating alzheimer's disease by administration of protective protein/cathepsin A

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMM., 1976, Vol. 72, No. 2, KAZAKOVA et al., "Crystallization of Cathepsin D", pages 747-752. *
FUSEK et al., Purification and Crystallization of Human Cathepsin D, Vol. 226, 1992, pages 555-557. *
RUDENKO et al., Three-dimensional Structure of the Human 'Protective Protein': Structure of the Precursor form Suggests a Complex Activation Mechanism, Vol. 3, No. 11, 15 November 1995, pages 1249-1259. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009148A1 (fr) * 1997-08-13 1999-02-25 Vertex Pharmaceuticals Incorporated Cristaux de l'helicase du virus de l'hepatite c ou des fragments de celle-ci comprenant une poche de fixation de l'helicase
US6183121B1 (en) 1997-08-14 2001-02-06 Vertex Pharmaceuticals Inc. Hepatitis C virus helicase crystals and coordinates that define helicase binding pockets
WO1999019465A1 (fr) * 1997-10-14 1999-04-22 E.I. Du Pont De Nemours And Company Trihydroxynaphtalene reductase: procedes permettant la determination de sa structure tridimensionnelle et la mise au point rationnelle de ses inhibiteurs
WO1999040182A3 (fr) * 1998-02-04 1999-10-07 Immunex Corp Enzyme de conversion cristalline du facteur tnf alpha et ses utilisations
US6842704B2 (en) 1998-02-04 2005-01-11 Immunex Corporation Crystalline TNF-α-converting enzyme and uses thereof
US8002891B2 (en) 1998-05-04 2011-08-23 Vertex Pharmaceuticals Incorporated Crystallization of C-Jun N-Terminal Kinase 3 (JNK3)
US7383135B1 (en) 1998-05-04 2008-06-03 Vertex Pharmaceuticals Incorporated Methods of designing inhibitors for JNK kinases
US6988041B2 (en) 2000-01-31 2006-01-17 Pharmacia & Upjohn Company Crystallization and structure determination of Staphylococcus aureus NAD synthetase
WO2001055443A1 (fr) * 2000-01-31 2001-08-02 Pharmacia & Upjohn Company Cristallisation et determination de la structure de staphylococcus aureus nad synthetase
WO2002020804A1 (fr) * 2000-09-08 2002-03-14 Prozymex A/S Structure de cristal peptidase dipeptidyl i et son utilisation
US7736875B2 (en) 2000-09-08 2010-06-15 Prozymex A/S Dipeptidyl peptidase I crystal structure and its uses
US6964867B2 (en) 2001-03-16 2005-11-15 Irm, Llc Method and apparatus for performing multiple processing steps on a sample in a single vessel
US6869792B2 (en) 2001-03-16 2005-03-22 Irm, Llc Method and apparatus for performing multiple processing steps on a sample in a single vessel
WO2002075277A3 (fr) * 2001-03-16 2003-02-20 Irm Llc Procede et appareil permettant d'effectuer des etapes de traitement multiples sur un echantillon dans une cuve unique
US7273716B2 (en) 2003-04-25 2007-09-25 Gilead Sciences, Inc. Methods and compositions for identifying therapeutic compounds with GS-7340 ester hydrolase
WO2005047898A3 (fr) * 2003-10-24 2005-09-15 Gilead Sciences Inc Procedes et compositions pour l'identification des composes therapeutiques
US7273715B2 (en) 2003-10-24 2007-09-25 Gilead Sciences, Inc. Methods and compositions for identifying therapeutic compounds with GS-9005 ester hydrolase A
US7273717B2 (en) 2003-10-24 2007-09-25 Gilead Sciences, Inc. Methods and compositions for identifying therapeutic compounds with GS-9005 ester hydrolase B
US9399791B2 (en) 2011-08-31 2016-07-26 St. Jude Children's Research Hospital Methods of treating alzheimer's disease by administration of protective protein/cathepsin A
US9840727B2 (en) 2011-08-31 2017-12-12 St. Jude Children's Research Hospital Methods and compositions to detect the level of lysosomal exocytosis activity and methods of use
US10533208B2 (en) 2011-08-31 2020-01-14 St. Jude Children's Research Hospital Methods of treating dementia associated with Alzheimer's disease with protective protein/cathepsin A (PPCA)

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