PRODUCTION AND USE OF MODIFIED CYSTATINS

Abstract: Cystatins that have been modified by glycosylation in order to enhance stability and activity are disclosed, as are methods of making such cystatins and methods of using such cystatins to inhibit proteolysis.

Description: PRODUCTION AND USE OF MODIFIED CYSTATINS FIELD OF THE INVENTION The present invention relates to protease inhibitors, specifically to cystatins, that have been modified by glycosylation in order to enh,ance stability and activity; to methods of making such modified protease inhibitors and to methods of using such modified protease inhibitors to inhibit proteolysis of a protein substrate. BACKGROUND Proteases are enzymes that degrade proteins. Proteases are classified by the substrate upon which they act and include serine proteases, cysteine proteases, aspartate proteases and metalloproteinases. Serine and cysteine proteases are widespread and are found in diverse organisms including eukaryotic and prokaryotic animals and plants. Cysteine proteases are generally well characterized enzymes having a known primary structure composed of alpha helices and beta pleated sheets (8). Proteases mediate many processes that are harmful to man, either by producing pathology or by causing economic loss, for instance by degrading foods. Protease-mediated pathology is known to be caused by a wide variety of org,anisms including bacteria, such as staphylococci and streptococci, fungi, arthropods, nematodes, protozoa such as amoebas, intestinal flagellates, haemoflagelates, such as Leishmania and trypanosomes and helminths. Proteases are known to be important in the pathology of certain viruses (9, 11 , 12, 31) including Polio virus, Herpes virus, Corona virus, HIV and Rotavirus. Proteases are also known to play a role in various diseases with no clear etiological agent, such as muscular dystrophy (7) and cancers (1) including breast cancer (2), and amyloid angiopathy, a genetic disease that often leads to fatal cerebral hemorrhages in young adults (10, 13). Proteases are also responsible for the spoilage of economically important foodstuffs, necessitating huge annual expenditures on preventative measures. For instance, the fungus Botrytis cinera causes widespread disease in over thirty species of commercial crop plants. Molds and fungi are the major destroyers of citrus fruit crops. Foods rich in protein, such as meat and fish, are degraded and made inedible by proteases from Pseudomonads and other bacteria. Foods with a high muscle content may be quickly broken down by endogenous proteases released from tissues upon death. These enzymes degrade myosin, and destroy the texture of the food. An important example of such spoilage occurs during the processing of surimi, which is a form of processed minced fish, commonly made from Pacific Whiting (Merluccius productus), and is the main ingredient of seafood analogs such as "imitation crab meat". Surimi is an important source of relatively cheap, high quality, low fat protein important to the diet of many people in the Far East and of increasing economic significance worldwide. Endogenous proteolytic enzymes released during surimi production cause rapid degradation of muscle tissue and lead to poor quality surimi (3, 4, 5, 6, 11 , 17, 18, 19). It is thought that the protease released from the fish tissue is a cathepsin, which is a common cysteine protease (3). Because of the role of proteases in these various processes, protease inhibitors have been investigated for their potential role in preventing disease and degradation of foodstuffs (4, 5, 18). Partially refined substances that contain protease inhibitors are commonly used in food processing, for instance, to prevent proteolytic breakdown of fish protein during the production of surimi (4, 17, 18). The most commonly used food-grade protease inhibitors are beef plasma protein (BPP), egg white powder and potato powder (4). Genetic engineering techniques have been used to introduce protease-inhibitor genes from chickens into cereal and grass plants to control protease- producing plant pathogens. Likewise, a plant protease inhibitor gene, from Cowpea, has been recombinantly introduced into tobacco, tomato, cotton and other plants to inhibit destruction by nematode worms (16). Cystatins are cysteine protease inhibitors that are members of Family 2 of the cystatin superfamily, characterized by a single chain of about 115 to 122 amino acids with a molecular weight of about 13000, having two disulfide bonds (7, 8, 10). Cystatins .and protease enzymes such as cathepsins form tight (but reversible) enzyme-inhibitor complexes with dissociation constants typically in the nannomolar range (10). A number of cystatins have been characterized including human cystatins (C, S, SN, SA, D, M and E), mouse cystatin, egg-white cystatin, bovine cystatin, carp cystatin, trout cystatin and salmon cystatin. In their natural state, cystatins protect the body by inhibiting the potentially harmful effects of proteolysis, and may prevent destruction of connective tissue by protease enzymes, for instance, lysosomal proteases, released from dying or damaged cells (16). Cystatins have been investigated for potential medical applications, for instance, to inhibit replication and pathology of Picornaviruses (12), Coronaviruses (9) and Herpes Simplex type 1 virus (15, 16, 30). Cystatins may also play a natural role in prevention of bacterial infection by E. coli, Shigella (13), Leishmania, Schistosoma and Entamobea (10) which appear to use proteases to facilitate tissue invasion. Cystatins are likely the primary protease inhibitors in food-grade protease inhibitor preparations such as beef plasma protein and egg white powder. Since cystatins are themselves proteins, they are prone to denaturation and loss of activity when exposed to unfavorable temperatures or pH. Many food production processes, including surimi production, involve elevated temperatures (17). Presently, in order to maintain cystatin activity, more cystatin must be added after cooling. Adding additional cystatin is both labor-intensive and expensive. Also, when cystatins are used for medical treatment, either as a topical or ingested medication, it is preferable for the cy statin-containing composition to be sterile. A common method of sterilization involves treatment with elevated temperatures. A cystatin that could maintain activity despite exposure to elevated temperatures would thus be useful in food processing and in drug formulation. SUMMARY OF THE DISCLOSURE The present invention provides modified, glycosylated, heat-stable cystatins and methods of making and using these cystatins. The present invention also provides nucleic acid molecules encoding such cystatins. The nucleic acid molecules of the invention have been modified so that when such a nucleic acid molecule is expressed in a eukaryotic cell, certain amino-acid residues of the expressed cystatin protein are glycosylated during post-translational modification of the protein. The resulting mature protein has attached, at specific amino acid residues, sugar molecule chains of varying length. The present invention includes the nucleic acid molecules that encode modified cystatins based on the cystatins from humans (C, S, SN, SA, D, M and E), egg white, cow, carp, trout and salmon. Various residues in the cystatin primary amino acid sequence have been identified where the introduction glycosylated residues increases heat stability of the expressed protein without severely affecting enzymatic activity. In human cystatin C, for instance, the sites for glycosylation include amino acid residues at positions 35, 36 and 79. The present invention also includes a method of making modified heat-stable cystatins by modifying the nucleic acid molecules that encode cystatins. Such nucleic acid molecules are modified at certain defined sites .and expressed in a eukaryotic cells. The present invention also includes a cell that contains at least one nucleic acid molecule encoding at least one modified, glycosylated, heat-stable cystatin. The cells of the invention may be of many types, for instance they may be cells from a yeast, a mammal, an insect, or a plant. The invention also includes methods of inhibiting proteolysis of a protein substrate by contacting the protein substrate with a modified heat-stable cystatin having at least one engineered glycosylation site. Such a method may be applied, for example, to food processing, such as the production of surimi. The invention also includes a method of treating a protease-mediated pathology of an organism, such as a mammal, a fish or a plant by administering to the organism a modified heat-stable cystatin of the invention. By such administration, the modified heat-stable cystatin contacts the protease that mediates the pathology, thereby inhibiting proteolysis by the protease and thereby treating the pathology. SEQUENCE LISTING SEQ. I.D. No. l shows the cDNA sequence and the amino acid sequence of native human cystatin C. SEQ. I.D. No.2 shows the amino acid sequence of native human cystatin C. SEQ. I.D. No.3 shows the cDNA sequence and the amino acid sequence of native human cystatin S. SEQ. I.D. No.4 shows the amino acid sequence of native human cystatin S. SEQ. I.D. No.5 shows the cDNA sequence and the amino acid sequence of native human cystatin SN. SEQ. I.D. No.6 shows the amino acid sequence of native human cystatin .SN. SEQ. I.D. No. 7 shows the cDNA sequence and the amino acid sequence of native human cystatin SA. SEQ. I.D. No. 8 shows the amino acid sequence of native human cystatin SA. SEQ. I.D. No. 9 shows the cDNA sequence and the amino acid sequence of native human cystatin D. SEQ. I.D. No. 10 shows the amino acid sequence of native human cystatin D. SEQ. I.D. No. 11 shows the cDNA sequence and the amino acid sequence of native human cystatin M. SEQ. I.D. No. 12 shows the amino acid sequence of native human cystatin M. SEQ. I.D. No. 13 shows the cDNA sequence and the amino acid sequence of native human cystatin E. SEQ. I.D. No. 14 shows the amino acid sequence of native human cystatin E. SEQ. I.D. No. 15 shows the cDNA sequence and the amino acid sequence of native egg white cystatin. SEQ. I.D. No. 16 shows the amino acid sequence of native egg white cystatin. SEQ. I.D. No. 17 shows the cDNA sequence and the amino acid sequence of native carp cystatin. SEQ. I.D. No. 18 shows the amino acid sequence of native carp cystatin. SEQ. I.D. No. 19 shows the cDNA sequence and the amino acid sequence of native salmon cystatin. SEQ. I.D. No. 20 shows the amino acid sequence of native salmon cystatin. SEQ. I.D. No. 21 shows the cDNA sequence and the amino acid sequence of native trout cystatin. SEQ. I.D. No. 22 shows the amino acid sequence of native trout cystatin. SEQ. I.D. No. 23 shows the cDNA sequence and the amino acid sequence of native bovine cystatin. SEQ. I.D. No. 24 shows the amino acid sequence of native bovine cystatin. SEQ. I.D. No. 25 shows the first of four oligonucleotides used to create a nucleotide coding for modified human cystatin C. SEQ. I.D. No. 26 shows the second of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C. SEQ. I.D. No. 27 shows the third of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C. SEQ. I.D. No. 28 shows the fourth of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C. SEQ. I.D. No. 29 shows a forward primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C. SEQ. I.D. No. 30 is a reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C. SEQ. I.D. No. 31 is the forward primer used in site-directed mutagenesis to introduce a glycosylation site at residue 36 of a modified human cystatin C. SEQ. I.D. No. 32 is the reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 36 of a modified human cystatin C. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the native amino acid sequence of the mature human cystatin C peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 2 shows the native amino acid sequence of the mature human cystatin S peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 3 shows the native amino acid sequence of the mature human cystatin SN peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 4 shows the native amino acid sequence of the mature human cystatin SA peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 5 shows the native amino acid sequence of the mature human cystatin D peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig.6 shows the native amino acid sequence of the mature human cystatin M peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 7 shows the native amino acid sequence of the mature human cystatin E peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 8 shows the native amino acid sequence of the mature Egg White cystatin peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 9 shows the native amino acid sequence of the mature bovine cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 10 shows the native amino acid sequence of the mature carp cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 11 shows the native amino acid sequence of the mature trout cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. Fig. 12 shows the native amino acid sequence of the mature chum salmon cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence. DESCRIPTION OF THE INVENTION A. Definitions A protein is said to be modified when it has been intentionally, artificially altered from its naturally occurring, wild-type form, e.g. , when the primary amino acid sequence of a cystatin protein has been intentionally, artificially altered to add a non-native glycosylation site somewhere in the protein. A DNA sequence is said to be modified when it has been intentionally, artificially altered from its naturally occurring, wild-type form, e.g., when the nucleotide coding for a cystatin protein has been mutated by site-directed mutagenesis to create a non-native nucleotide addition, deletion of substitution. The term engineered may be used synonymously with the term modified. To say that an organism, or nucleotide sequence has been genetically engineered means that it has been intentionally, artificially genetically altered from its naturally occurring, wild-type genetic form, for instance, a DNA sequence is said to be genetically engineered when its sequence has been intentionally, artificially genetically altered from its wild-type sequence. Any genetically engineered organism or nucleotide sequence has been modified. Heat stability refers to the ability of a protein to function at high temperatures. Proteins typically lose activity as the temperature is raised above their normal in vivo operating range, and generally become denatured as the temperature increases further. A modified protein may be more heat stable than the unmodified form of the protein, meaning that the modified form of the protein retains greater activity at higher temperatures than the unmodified form of the protein. To say a feature has been introduced means that a non-wild-type feature has been intentionally, artificially added, for inst. nce, a glycosylation site may be said to be introduced into a protein if it has been intentionally, artificially added to the protein, also a nucleotide may be said to be introduced into a cell or other nucleotide sequence when it has been intentionally, artificially added into a cell or sequence, likewise, a deletion may be introduced into a DNA sequence. Likewise, amino acid substitutions, additions and deletions may be introduced into a protein. A glycosylation site is a place on a molecule at which sugars may be added. The addition of sugars (glycosylation) may occur at introduced sites in modified proteins such as at amino acid residue 37 of the modified human cystatin C. A glycosylation site in a protein is denoted by the general formula X (#) Z, where X = the native amino acid that has been removed, and # = the position of that amino acid in the protein, and Z = the substituted amino acid that has been inserted in place of X. For instance, Ala (37) Ser denotes that Alanine at position 37 has been removed and replaced with Serine; likewise, for one-letter code, A (31) N denotes that Alanine at position 31 has been removed and replaced with Asparagine. The numbering system of cystatin proteins in this text reflects that shown in the accompanying figures. While alternative numbering systems are possible, the nomenclature use herein is intended to identify a particular amino acid residue, rather than any residue that happens to be at a given distance from an arbitrary point on an amino acid sequence. An organism refers to any organism of any kingdom, phylum, class, order, family, genus or species. A pathology refers to a state that is measurably or detectably at variance with normal, healthy, non-disease-state physiology. A cancer refers to any neoplastic transformation or any tissue that has undergone neoplasia and includes solid, non-solid, benign and malignant transformed tissue of both plants and animals. A protease-mediated pathology is any pathology that is caused in part or in whole by the action of a protease, for instance, the proteolytic invasion of human tissue by a cancer cell, the proteolytic destruction of human tissue by a bacterium or a protozoan (e.g., Pseudomonas or Leishmaniά) and the tissue destruction of a plant by a fungus (e.g., Phytopthora infestans) are all examples of protease-mediated pathologies . To say that a polynucleotide (or a gene or genome) is recombinant means that it has been altered by the addition at some site of non-native nucleic acids or nucleotides, i.e. , nucleotides that are not normally found in the particular polynucleotide, or that are not normally found at that site. For instance, a human cystatin gene that has been altered by nucleotide substitution is said to be recombinant. Likewise, a recombinant protein is a protein that is the product of a recombinant polynucleotide and that contains non-native amino acid residues. An isolated nucleic acid has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA. The term isolated thus encompasses nucleic acids purified by standard nucleic acid purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. Nucleic acid probes and primers may readily be prepared based on the nucleic acid sequences provided by this invention. A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are well known in the field of molecular biology. Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. Probes and primers as used in the present invention preferably comprise at least 15 nucleotides of the nucleic acid sequences that encode a modified cystatin protein. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise 20, 30 or 40 consecutive nucleotides of the disclosed nucleic acid sequences. Methods for preparing and using probes and primers are described in a number of reference works, for example Sambrook et al. (1989) (26); Ausubel et al., (1987) (25); Innis et al. , (1990) (27). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991 , Whitehead Institute for Biomedical Research, Cambridge, MA). A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. The term purified peptide does not require absolute purity, rather, it is intended as a relative term. Thus, for example, a purified cystatin preparation is one in which cystatin is enriched compared to the cystatin in its natural environment, i.e. , within a cell. A purified preparation of a cystatin is prepared such that the cystatin represents at least 20% of the portion of the protein content of the preparation. Preparations comprising at least 50% , 75% or at least 90% cystatin (expressed as a percentage of total protein) may be desirable for certain applications. B. General Methods The present invention utilizes standard laboratory practices for the cloning, manipulation and sequencing of nucleic acids, purification and analysis of proteins and other molecular biological and biochemical techniques, unless otherwise stipulated. Such techniques are explained in detail in standard laboratory manuals such as Sambrook et al., (1989)(25) and Ausubel et al., (1987)(26). (1) Production, cloning and expression of modified cystatins A modified nucleotide sequence that codes for a cystatin protein having at least one glycosylation site may be produced by making synthetic sequences using a commercial polynucleotide synthesizer. The synthetic nucleotides are designed on the basis of the known nucleotide sequence of the cystatin gene and the known codon usage for the cell type in which the nucleotides are to be expressed. A suitable target site for the introduction of a glycosylation site is identified based on the primary amino acid sequence of the cystatin to be modified. A nucleotide sequence is designed that will code for a peptide that includes such a site. The synthetic nucleotide sequence may then be synthesized as described herein. A series of anti-parallel and complementary nucleotides may be synthesized and annealed together to form synthetic double-stranded nucleotide fragment. These fragments may then be ligated together to produce a contiguous double-stranded nucleotide that codes for a particular cystatin having one or more engineered glycosylation sites. The synthetic nucleotides may be amplified in vitro using the Polymerase Chain Reaction (PCR) (27) so that many copies of the nucleotide are available for cloning into a prokaryotic cloning vector (26) or into an expression vector, as explained below. Additional glycosylation sites may be introduced using site-directed mutagenesis to add, delete or substitute particular amino acid residues. Various standard techniques are known to carry out site-directed mutagenesis (26, chapter 15), and commercial kits are also available such as the QUICKCHANGE™ mutagenesis site-directed mutagenesis kit (STRATAGENE™, CA). Nucleotides may be modified or synthesized so as to include restriction enzyme sites that can be used for cloning. The modified nucleotide may be cloned into a standard prokaryotic cloning vector, for example pBR 322, pUCIS or pUC!9 (26, chapter 1). The sequence of the cloned nucleotide may be checked by sequencing using standard methods (26, chapters 1 and 13). Modified nucleotides may be cloned into an expression vector that allows protein production in a particular cell type. Since the proteins of the invention are glycosylated, it is required that the cell type in which the proteins are expressed can readily carry out post-translational modification including glycosylation. Typically, a eukaryotic cell is used that glycosylates peptides at the Asn- X-Ser/Thr motif. Yeast cells are commonly used for such a purpose. Standard cloning techniques may be used (26, chapter 9). Such expression vector/cell systems are well known and commercially available and include vector/cell combinations that carry out post-translational modifications required for the proper expression of glycosylated eukaryotic proteins. Various yeast strains and yeast-derived vectors are commonly used for this type of expression, for instance, Pichia pastoris expression systems that may be used to practice the present invention may be obtained from INVITROGEN™. Such systems include suitable Pichia pastoris strains, vectors, reagents, transformants, sequencing primers and media. Available strains include a GS115 his 4 deficient strain, a KM71 aoxl deficient strain, a GS115 His + Mut " strain for extracellular expression and a His + Mut " strain for intracellular expression (33). Non-yeast eukaryotic vectors may equally be used for expression of the modified nucleotides. Mammalian vector / host cell systems that contain genetic and cellular control elements capable of carrying out transcription, translation and post-translational modification are well known in the art. Examples of such systems are the well known Baculovirus system, the Ecdysone-inducible mammalian expression system that uses regulatory elements from Drosophila melanogaster to allow control of gene expression, and the Sindbis viral expression system that allows high level expression in a variety of mammalian cell lines, which are available from INVITROGEN™. The cloned expression vector may then be transformed into a particular cell type and the nucleotide expressed. Many different types of cell may be used to express the modified nucleic acid molecules. Examples of such cells include cells of yeasts, fungi, insects and humans and plants, including transformed and non-transformed cells. For instance, common mammalian cells that could be used for the invention include human HeLa cells, SW-527 human cells (ATCC deposit #7940), WISH cells (ATCC deposit #CCL-25), Daudi cells (ATCC deposit #CCL-213), Mandin- Darby bovine kidney cells (ATCC deposit #CCL-22) and Chinese Hamster ovary cells (ATCC deposit #CRL-2092). Common yeast cells include Pichia pastoris (ATCC deposit #201178) and Saccharomyces cerevisiae (ATCC deposit #46024). Insect cells include cells from Drosophila melanogaster (ATCC deposit #CRL-10191), the cotton bollworm (ATCC deposit #CRL-9281) and from Trichoplusia ni egg cell homoflagelates. Fish cells that may be used include those from rainbow trout (ATCC deposit #CLL-55), salmon (ATCC deposit #CRL-1681) and Zebrafish (ATCC deposit #CRL-2147). Amphibian cells that may be used include those of the Bullfrog, Rana catesbelana (ATCC deposit #CLL-41). Reptile cells that may be used include those from Russell's Viper (ATCC deposit #CCL-140). Plant cells that could be used include Chlamydomonas cells (ATCC deposit #30485), Arabidopsis cells (ATCC deposit #54069) and tomato plant cells (ATCC deposit #54003). Many of these cell types are commonly used and are available from the ATCC as well as from commercial suppliers such as PHARMACIA™ and INVITROGEN™. Expressed protein may be accumulated within a cell or may be secreted from the cell. Such expressed protein may then be collected and purified. This protein may then be characterized for activity and heat stability and may be used to practice the methods of the invention. The amino acid sequences of the cystatins (figures 1-12) are shown in their mature form without a signal peptide. The signal peptide is cleaved off during post-translational modification to produce the mature peptide. The invention may be equally practiced with cystatin peptides which retain the signal peptide. (2) Measurement of protease activity and heat resistance Protease inhibition activity of modified cystatins is assayed by measuring the reduction in activity of the protease papain. A substrate is used that releases nitroaniline into the reaction medium. The amount of nitroaniline released is determined by measuring light absorption by the solution. This assay is described in detail in Example 4, below. Heat resistance of the modified cystatin is determined by heating a solution containing a known amount of modified cystatin, cooling it, and adding it to a mixture containing a known amount of protease and a known amount of protein. Protease activity is measured by determining turbidity of the mixture after a set time and also by using the nitroaniline assay as described below. (3) Methods of using modified cystatins The modified cystatins of the invention may be used to inhibit proteolysis of a protein substrate in generally the same way as non-modified cystatins are used. Such uses include inhibition of proteolysis in food processing (4), therapeutic treatment of viral disease such as those caused by Herpes Simplex (11 , 30), picornaviruses (20) and coronavirus (9). The present invention includes a general method of inhibiting proteolysis of a substrate by providing the modified cystatin and by contacting this cystatin with the substrate. Such a substrate may be any substrate containing protein. Such a modified cystatin may be used in food processing applications, in agricultural applications and in human and non-human medical applications. For instance, a protein substrate such as minced Pacific Whiting (as used for surimi production) may be treated with a modified cystatin of the invention to inhibit proteolysis caused by the release of endogenous protease from the fish tissue. Suggested concentrations for the application of modified cystatin are, for instance, for surimi processing, from about lμg/g to lOOμg/g of surimi. The present invention also includes methods of using a modified cystatin for the inhibition of proteases for therapeutic purposes. For instance, the cystatins of the invention may be used to inhibit tissue destruction and invasion by pathogens such as staphylococci and streptococci. Streptococcus is the etiological agent of the common skin disease impetigo, and certain particularly rapacious "flesh eating" strains of Staphylococcus and Streptococcus have recently received much media attention, particularly because of their multiple antibiotic resistance. The effectiveness of cystatins of the invention in preventing bacterial invasion of host-tissues can be measured, for example, by the method of Betts and Finlay (32). This method can be used to determine tissue invasion of green monkey kidney cells (ATCC deposit #CCL-70) by bacteria such as E. coli and Salmonella typhimurium. For medical applications the modified cystatin may be administered topically or systemically. The cystatin may be formulated with a carrier or pharmacologically acceptable excipient. For instance, the cystatin may be mixed with a carrier such as a petroleum-based or lanolin-based oil to form a gel, and administered topically at the site of infection, thereby contacting the modified cystatin with the protease and preventing tissue destruction. The cystatins of the present invention may also be administered systemically, either orally, intravenously, sub-cutaneously, transdermally or by other methods. For instance, the cystatin of the present invention may be formulated into a tablet or solution form and administered orally. Formulation of drugs into pills and tablets is well known in the art. Systemic administration may be used to treat infections that cannot be treated topically such as cancer and systemic viral infections of humans and animals. For topical medical treatment, concentrations of modified cystatin in a cream or ointment may be from about lng to 100 mg per gram total weight of ointment, or may be from about lμg to 1 mg total weight, or may be from about lOμg to lOOμg total weight. For ingested medications, the amount of modified cystatin per Kg mass of a patient may be from about lng to 100 mg, or may be from about lμg to 1 mg, or may be from about lOμg to lOOμg. The cystatins of the present invention may be used to inhibit proteolysis caused by pathogens of crop plants and fruits. For instance, the modified cystatins may be applied to the surface of fruit or crops to inhibit proteases produced by plant pathogens such as fungi, for instance, Botrytis. The modified cystatin may, for instance, be sprayed or painted onto crops, either in a pure form or diluted in a carrier liquid such as water. The cystatins of the invention show superior heat resistance, maintaining a high degree of activity after exposure to elevated temperatures. These heat resistant cystatins may be particularly useful where sterility is required, such as in medical applications where it is generally desirable for medicines to be uncontaminated with biological organisms. For instance, the cystatins of the present invention may be formulated into topical ointments or pills which may then be packaged and sterilized and administrated therapeutically. Also, food processing, such as surimi production, involves elevated temperatures. The cystatins of the invention are useful in such processes due to their enhanced resistence. C. Production of Modified Human Cystatin C Glycosylation of one or more amino acid residues at specific sites in the human cystatin C peptide increases heat stability of the cystatin without substantially inhibiting functionality. Glycosylation at other, inappropriate sites, may drastically decrease or destroy the protein's inhibitory function. The invention identifies three amino acid residues of human cystatin C that may be modified to introduce a glycosylation site; one or more of these sites may be modified to produce a cystatin protein having superior properties, such as enhanced heat stability. The amino acid sequences for native and modified human cystatin C are shown in Fig. 1. Amino acid substitutions to produce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more of these amino acid substitutions may be present in a modified cystatin protein of the present invention. The cDNA sequence for native human cystatin C is shown in Seq. ID No.1. Human cystatin C protein may be modified to introduce glycosylation sites (Asn-X-Ser/Thr) at amino acid residues 35, 36 or 79 by the introduction of amino acids substitutions at positions 37, 36 or 81, respectively. One or more of the following specific substitutions may be made: A (37) S, A (37) T, K (36) N, D (81) S, D (81) T. It should be noted that the peptide of cystatin C in fig. 1 is shown in its mature form without a signal peptide. The signal peptide is cleaved off during post-translational modification to produce the mature peptide. The invention may be equally practiced with cystatin peptides which retain the signal peptide. The same is so for all the other cystatins, figs. 2 -12. D. Production of Modified Human Cystatin S The amino acid sequences for native and modified human cystatin S are shown in Fig. 2. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin S is shown in Seq. ID No. 3. The human cystatin S protein may be modified by the introduction of the following amino acid substitutions: A (31) N, K (37) N, A (38) S, A (38) T, L (81) N, D (82) T, and D (82) S. E. Production of Modified Human Cystatin SN The amino acid sequences for native and modified human cystatin SN are shown in Fig. 3. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin SN is shown in Seq. ID No. 5. The human cystatin SN protein may be modified to introduce one or more glycosylation sites by the introduction of one or more of the following amino acid substitutions: A (31) N, A (38) S, A (38) T, K (37) N, L (81) N, D (82) S, D (82) T. F. Production of Modified Human Cystatin SA The amino acid sequences for native and modified human cystatin SA are shown in Fig. 4. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin SA is shown in Seq. ID No. 7. The human cystatin SA protein may be modified by the introduction of the following amino acid substitutions: V (31) N, A (38) S, A (38) T, K (37) N, D (82) S, D (82) T, L (81) N. G. Production of Modified Human Cystatin D The amino acid sequences for native and modified human cystatin D are shown in Fig. 5. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin D is shown in Seq. ID No. 9. The human cystatin D protein may be modified by the introduction of the following amino acid substitutions: A (31) N, V (38) S, V (38) T, D (42) S, D (42) T, D (83) S, D (83) T, P (86) S, P (86) T, Q (90) S, Q (90) T, Y (44) N. H. Production of Modified Human Cystatin M The amino acid sequences for native and modified human cystatin M are shown in Fig. 6. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin M is shown in Seq. ID No. 11. The human cystatin M protein may be modified by the introduction of the following amino acid substitutions: V (35) N, M (40) N, G (41) S, G (41) T, S (42) N, I (45) S, I (45) T, R (78) N, R (81) N, D (88) N, L (89) N. I. Production of Modified Human Cystatin E The amino acid sequences for native and modified human cystatin E are shown in Fig. 7. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin E is shown in Seq. ID No. 13. The human cystatin E protein may be modified by the introduction of the following amino acid substitutions: V (28) N, M (33) N, G (34) S, G (34) T, S (35) N, I (38) S, I (38) T, D (81) N, L (82) N. J. Production of Modified Egg White Cystatin The amino acid sequences for native and modified egg white cystatin are shown in Fig. 8. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native egg white cystatin is shown in Seq. ID No. 15. The egg white cystatin protein may be modified by the introduction of the following amino acid substitutions: A (35) S, A (35) T, R (34) N, K (39) S, K (39) T, K (39) N, Y (40) N, L (78) N, K (91) N, Y (92) N. K. Production of Modified Bovine Cystatin The amino acid sequences for native and modified bovine cystatin are shown in Fig. 9. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native bovine cystatin is shown in Seq. ID No. 23. The bovine cystatin protein may be modified by the introduction of the following amino acid substitutions: A (29) N, R (36) S, R (36) T, K (35) N, A (40) S, A (40) T, Y (41) N, L (79) N, D (80) S, D (80) T, P (88) S, P (88) T. L. Production of Modified Carp Cystatin The amino acid sequences for native and modified carp cystatin are shown in Fig. 10. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native carp cystatin is shown in Seq. ID No. 17. The carp cystatin protein may be modified by the introduction of the following amino acid substitutions: Q (31) S, Q (31) T, G (30) N, A (35) S, A (35) T, K (39) N, K (91) N. M. Production of Modified Trout Cystatin The amino acid sequences for native and modified trout cystatin are shown in Fig. 11. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native trout cystatin is shown in Seq. ID No. 21. The trout cystatin protein was modified by the introduction of the following amino acid substitutions: K (29) N, K (30) S, K (30) T, M (34) T, M (34) S, K(39)N, K (88) N. N. Production of Modified Chum Salmon Cystatin The amino acid sequences for native and modified chum salmon cystatin are shown in Fig. 12. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native chum salmon cystatin is shown in Seq. ID No. 19. The chum salmon cystatin protein may be modified by the introduction of the following amino acid substitutions: K (29) N, K (30) S, K (30) T, M (34) S, M (34) T, K (88) N. EXAMPLE 1 Synthesis of nucleotide sequences that code for modified human cystatin C Cloning was done using Uιe pUCl9 cloning vector and E.coli using standard gene cloning techniques (26). The yeast strain Pichia pastoris KM71 was used to express mammalian genes and constructs. T4 DNA ligase, restriction enzymes, the 7-DEAZA sequencing kit and blunting kit were all purchased from TAKARA SHUZO™ of Kyoto, Japan. The oligonucleotide in vitro mutagenesis system (version 2) was purchased from AMERSHAM™ International. CM TOYOPEARL™ 650M resin was purchased from TOSOH™ of Tokyo. Concanavalin A-sepharose and α-methylm.annoside were purchased from PHARMACIA™ and from WAKO™ of Tokyo, respectively. Sephadex-G50 was purchased from PHARMACIA™. M 13m/? 19 was used as a vector for cDNA construction. The modified nucleotide sequences were made in two steps. First, a synthetic double-stranded DNA was constructed that codes for human cystatin C, modified to have a glycosylation site at residue number 79. This DNA was made by chemically synthesizing four oligonucleotides using an automated oligonucleotide synthesizer (SEQ. I.D. No. 25, SEQ. I.D. No. 26, SEQ. I.D. No. 27 and SEQ. I.D. No. 28). The oligonucleotides were chosen on the basis of the known nucleotide sequence of the native human cystatin C gene and the known codon usage of P. pastoris (Table 1). DNA was sequenced using the Sanger method (26, chapter 13). Polypeptides were sequenced using the Edman degradation method in a gas-phase protein automated sequencer (SHIMADZU™ model PSQ2). Glycine GGT or GGA Glutamic acid GAG of GAA Aspartic acid GAC or GAT Valine GTT or GTC Alanine GCT or GCC Arginine AGA or CGT Serine TCT or TCC Lysine AAG Asparagine AAC Methionine ATG Isoleucine ATT or ATC Threonine ACT or ACC Tryptophan TGG Cysteine TGT Tyrosine TAC Leucine TTG or CTG Phenylalanine TTC Glutamine CAA or CAG Histidine CAC or CAT Proline CCA or CCT TABLE 1 The complementary pairs of the four oligonucleotides were annealed together and the resulting double-stranded fragments were ligated using T4 ligase. The resulting synthetic open reading frame contained an Xhol site at the 5' end and an Xba site at the 3' end that were used for cloning. The gene product was ligated into pUCl9 (26, chapter 1) and sequenced in both directions to check that the sequence was as predicted. Second, an N-glycosylation site was introduced at either residue 35 or 36 using the QUICKCHANGE™ site-directed mutagenesis kit (STRATAGENE™, CA) according to manufacturers instructions and using the forward and reverse primers shown in SEQ. I.D. Nos. 29- 32. EXAMPLE 2 Transformation and clone selection for production of modified human cystatin C The yeast expression plasmids pYG-100 (20) and pPICZ α-C containing Saccharomyces cerevissiae α factor secretion signal and alcohol oxidase (AOX1) gene promoter were used to express the proteins of the invention. P. pastoris strain KM71 was transformed using the Pichia EASYCOMP™ transformation system (INVITROGEN™, CA). Zeocin-resistant transformants were selected from yeast extract peptone dextrose sorbitol medium (YPDS) agar plates containing the zeocin. PCR sequencing was used to confirm insertion of the cystatin C gene in Pichia clones. EXAMPLE 3 Expression and purification of modified human cystatin C The Pichia transformants were incubated in yeast minimal medium (YMM). The Pichia transformants were grown at 30°C for one day in 5 mL of YMM, and then subcultured at 30°C for four days in 500 ml of fresh YMM. 100% mefhanol was added into the fresh YMM to a final concentration of 0.5% mefhanol every 24 hours to maintain induction of the cystatin gene. Recombinant modified human cystatin C was secreted in the Pichia culture media. The extracellular proteins were collected using an ultrafiltration system with 10,000 MW cut-off (PELLICON™ cassette filter, MILLIPORE™, Bedford, MA). The crude proteins thus recovered were applied to a column of Q-SEPH AROSE FAST FLOW™ (PHARMACIA™, Upsala, Sweden) equilibrated with a linear gradient of 0-0.5 M NaCl in 20 mM Tris-HCI buffer (pH7.5). The fraction including the cystatin was determined by the inhibitory activity against papain as described below. The fraction was applied to a column of sephacryl S-100 HR (PHARMACIA™) equilibrated with 0.15 M NaCl-20 mM Tris-HCI buffer (pH7.5). The fraction which showed the inhibitory activity was collected. EXAMPLE 4 Assay of inhibitory activity and heat resistance of modified human cystatin C Cystatin activity was assayed by measuring papain inhibitory activity using Nα- Benzoyl-DL-Arg-p-Nitroanilide (Bz-Arg-NA) (34). 0.1 ml of cystatin sample and 0.1 ml of papain solution (0.5 mg/ml) were pipetted into 0.1 mL of 50 mM Tris-HCI buffer (pH7.5) containing 100 mM Bz-Arg-NA, 2 mM EDTA, and 5 mM cystein. The solution was incubated for 25 min at 37°C. The reaction was stopped with 0.2 ml of 30% acetic acid, and the nitroaniline liberated by enzymatic activity is quantified by measurement of light absoφtion of the solution at 410nm. The inhibitory activity was expressed as the amount of enzyme (mg) inhibited by 1 mg of inhibitor (U/mg). Heat resistance assays were performed by heating a solution containing a known amount of cystatin to a controlled temperature for a controlled time; cooling the solution; mixing the cooled cystatin solution with a known amount of papain and protein substrate, and measuring turbidity of the mixture. The recombinant cystatins were heated to 95°C at a rate of l°C/min for 30°C in 50 mM sodium phosphate buffer (pH 7.5). Protein concentration was 1 mg/ml. At preset temperatures, each heated sample was transferred into a cuvette and the turbidity measured at 500 nm. The residual papain-inhibiting activity of the heated samples was also measured as described above. This procedure was repeated in triplicate. The above examples are provided by way of illustration only and are in no way intended to limit the scope of the invention. One of skill in the art will see that the invention may be modified in various ways without departing from the spirit or principle of the invention. We claim all such modifications. References 1. Keppler et al., (1993) In Proteases and Cancer Colloquium Queen's Univ. , Belfast, 22, 43-49 2. Kuopio et al. , (1998) Cancer Research 58 (3), 432-436 3. Seymour et al., (1994) Journal of Agricultural and Food Chemistry 42, 2421 4. Weerasinghe et al., (1996) 7. Agric. Food Chem. 44, 2584-2590 5. Izquierdo-Pulido et al., (1994) /. Agric Food Chem 42, 616-622 6. Yamashita et al. , (1991) Nippon Suisan Gakkaishi 57 (10), 1917-1922 7. Barrett et al. , The Biochemical Journal (1986) Letters 236 (1), 311-312 8. Turk et al., (1991) FEBS 285 (2) 213-219 9. Collins et al. , (1998) Oral Microbiol Immunol. 13 (1), 59-61 10. Barrett (1987) TIBS 12, 193-196 11. Grubb et al. , (1995) U.S. Patent No. 5,432,264 12. Korant et al., (1985) Biochem. and Biophys. Res. Comm. 127 (3), 1072-1076 13. Abrahamson et al., (1988) FEBS Letters 236 (1), 14-18 14. Nakamura et al , (1993) J. ofBiol. Chem. 268 (17), 12706-12712 15. Saitoh et al., (1998) Arch Biochem Biophys 352 (2), 199-206 16. Atkinson et al., (1996) PCT Patent No. WO 96/16173 17. An et al. , (1994) Journal of Food Science 59 (2), 277 18. Morrissey et al., (1993) Journal of Food Science 58 (5), 1050 19. An et al. , (1994) Journal of Food Science 59 (5), 1013 20. Turk et al., (1990) U.S. Patent No. 4,902,509 12. Yamashita et al., (1990) Nippon Suisan Gakkashi 56 (8) 1271-1277 22. Saeki et al. , (1995) Journal of Food Science 60 (5), 23. Yamashita et al., (1991) Comp. Biochem. Physiol. 100A (3) 749-751 24. Nakamura et al. , (1993) FEBS 328 (3) 259-262 25. Ausubel et al., (1987). Current Protocols in Molecular Biology, ed. Greene Publishing and Wiley-Interscience: New York (with periodic updates) 26. Sambrook et al. , (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1- 3, ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY 27. Innis et al., (1990). PCR Protocols: A Guide to Methods and Applications , Academic Press: San Diego 28. Nakamura et al., (1996) FEBS Letters 383 251-254 29. An et al., (1995) J. Agric. Food Chem. 43 327-330 30. Bjorck et al. , J. Virol. 64 (2) 941-943 31. Wyatt, R.G. et al. , (1980) Science, 207, 189-191 32. Betts et al., (1992) Can. J. Microb. 38 852-857 33. Invitrogen Product Catalogue, 1998. Invitrogen, Carlsbad, CA. 34. Barrett et al., (1981) Methods. Enzymol. 80: 771-778

Claims: CLAIMS What is claimed: 1. A modified cystatin selected from the group consisting of: a) modified human cystatin C having at least one of the following modifications: K (36) N A (37) S A (37) T D (81) S D (81) T b) modified human cystatin S having at least one of the following modifications: A (31) N K (37) N A (38) S A (38) T L (81) N D (82) S D (82) T c) modified human cystatin SN having at least one of the following modifications: A (31) N A (38) S A (38) T K (37) N L (81) N D (82) S D (82) T d) modified human cystatin SA having at least one of the modifications selected from the group consisting of: V (31) N A (38) S A (38) T K (37) N D (82) S D (82) T L (81) N e) modified human cystatin D having at least one of the modifications selected from the group consisting of: A (31) N V (38) S V (38) T D (42) S D (42) T V (44) N D (83) S D (83) T P (86) S P (86) T Q (90) S Q (90) T f) modified human cystatin M having at least one of the modifications selected from the group consisting of: V (35) N M (40) N G (41) S G (41) T S (42) N I (45) S I (45) T R (78) N R (81) N D (88) N L (89) N g) modified human cystatin E having at least one of the modifications selected from the group consisting of: V (28) N M (33) N G (34) S G (34) T S (35) N I (38) S I (38) T D (81) N L (82) N h) modified egg white cystatin having at least one of the modifications selected from the group consisting of: A (35) S A (35) T R (34) N K (39) S K (39) T K (39) N Y (40) N L (78) N K (91) N Y (92) N i) modified bovine cystatin having at least one of the modifications selected from the group consisting of: A (29) N R (36) S R (36) T K (35) N A (40) S A (40) T Y (41) N L (79) N D (80) S D (80) T P (88) T P (88) S j) modified caφ cystatin having at least one of the modifications selected from the group consisting of: Q (31) S Q (31) T G (30) N A (35) S A (35) T K (39) N K (91) N k) modified trout cystatin having at least one of the modifications selected from the group consisting of: K (29) N K (30) S K (30) T M (34) T M (34) S K (88) N 1) A modified chum salmon cystatin having at least one of the modifications selected from the group consisting of: K (29) N K (30) S K (30) T M (34) S M (34) T K (88) N 2. A nucleic acid molecule encoding a modified cystatin according to claim 1. 3. A cell comprising a nucleic acid molecule according to claim 2. 4. A cell according to claim 3 wherein the cell is selected from the group consisting of: a yeast cell, an insect cell, a human cell, a bacterial cell and a plant cell. 5. A method of inhibiting proteolysis of a protein substrate by contacting the cystatin protein substrate with the modified cystatin of claim 1. 6. A modified human cystatin C having at least one of the following modifications: K (36) N A (37) S A (37) T D (81) S D (81) T 7. A nucleic acid molecule encoding a modified cystatin according to claim 6. 8. A cell comprising a nucleic acid molecule according to claim 7. 9. A cell according to claim 8 wherein the cell is selected from the group consisting of: a yeast cell, an insect cell, a human cell, a bacterial cell and a plant cell. 10. A method of inhibiting proteolysis of a protein substrate by contacting said cystatin protein substrate with a modified cystatin according to claim 6.

Inventors: NAKAI SHURYO, NAKAMURA SOICHIRO, OGAWA MASAHIRO, NAKAI, SHURYO, NAKAMURA, SOICHIRO, OGAWA, MASAHIRO
Applicants: NAKAI SHURYO, NAKAMURA SOICHIRO, OGAWA MASAHIRO, UNIV BRITISH COLUMBIA, NAKAI, SHURYO, NAKAMURA, SOICHIRO, OGAWA, MASAHIRO, THE UNIVERSITY OF BRITISH COLUMBIA
Application year: 1999
Main classification: C12N15
IPC codes: C07K14, C12N1, C12N15