Leukocystatin, A New Class II Cystatin Expressed Selectively by Hematopoietic Cells
1998; Elsevier BV; Volume: 273; Issue: 26 Linguagem: Inglês
10.1074/jbc.273.26.16400
ISSN1083-351X
AutoresS Halfon, J. E. Ford, Jessica Foster, Lynette Dowling, Linda Lucian, Marissa Sterling, Yuming Xu, Mary C. Weiss, Mami Ikeda, Debra Liggett, Allison Helms, Christophe Caux, Serge Lebecque, Chuck Hannum, Satish Menon, Terrill K. McClanahan, Daniel M. Gorman, Gérard Zurawski,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoWe describe a new cystatin in both mice and humans, which we termed leukocystatin. This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions. As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs. In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns. The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAG™ fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins. The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system. We describe a new cystatin in both mice and humans, which we termed leukocystatin. This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions. As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs. In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns. The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAG™ fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins. The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system. Cysteine proteases play many very important roles in the immune system. For instance, the de-ubiquinating enzymes are cysteine proteases, whereas lysosomal proteases are involved in antigen presentation both through the degradation of proteins to antigenic peptides and by processing the invariant chain of class II major histocompatibility complexes (1Riese R.J. Wolf P.R. Bromme D. Natkin L.R. Villadangos J.A. Ploegh H.L. Chapman H.A. Immunity. 1996; 4: 357-366Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). However, the overexpression of these proteases can be detrimental to cells, as can their release into the extracellular space. Therefore, their activities in these cells are controlled by a variety of mechanisms, including the presence of macromolecular protease inhibitors.The cystatins make up a class of very tight, reversible, competitive inhibitors of the papain family of cysteine proteases. Cystatins have been divided into four classes based upon their sequences and properties. Class I, also called the stefins, are a group of intracellular proteins of approximately 100 residues that contain no disulfide bonds. Class II cystatins are secreted inhibitors of about 120 amino acids containing two disulfide bonds. Class III cystatins, known as the kininogens, contain three domains, each of which resembles Class II cystatins; two of these domains possess inhibitory activity. Finally, Class IV cystatins constitute a poorly understood group of glycoproteins with two nonfunctional cystatin domains. The amino acid sequences and genomic structures within each family are highly conserved. Cystatins are expressed throughout the body in a tissue-specific manner. Mutations in some cystatins or alterations in the balance of these with their cognate cysteine proteases have been implicated in several diseases (2Calkins C.C. Sloane B.F. Biol. Chem. Hoppe-Seyler. 1995; 376: 71-80PubMed Google Scholar, 3Henskens Y.M. Veerman E.C. Nieuw Amerongen A.V. Biol. Chem. Hoppe-Seyler. 1996; 377: 71-86Crossref PubMed Google Scholar). Many studies, involving changes of peptide sequence, have shown that three regions of the cystatin, which form a "wedge" that can associate with the active-site cleft, are all required for tight binding to the protease. These studies have been confirmed by the crystal structure of the cystatin B-papain complex (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar) and supported by other structural studies showing that chicken egg white cystatin, a Class II cystatin, has the same fold as the Class I cystatin B (5Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar, 6Dieckmann T. Mitschang L. Hofmann M. Kos J. Turk V. Auerswald E.A. Jaenicke R. Oschkinat H. J. Mol. Biol. 1993; 234: 1048-1059Crossref PubMed Scopus (81) Google Scholar).In this paper, we describe the characterization of a new Class II cystatin, leukocystatin, specifically expressed by hematopoietic cells. The unique features of the amino acid sequence suggest that the as yet unidentified target protease is not one of the commonly studied lysosomal cysteine proteases, although leukocystatin is an active inhibitor of these cathepsins. In addition, the unusual genomic structure of the mouse protein and the amino acid sequences of both the human and mouse inhibitors suggest that they are quite divergent from other Class II cystatins.DISCUSSIONWe have discovered a novel hematopoietic cell-specific Class II cystatin from an EST analysis of human dendritic cells. This protein, which we have called leukocystatin, has all the features of a Class II cystatin, but it has some notable characteristics. For example, leukocystatin contains lysine residues at two positions that are strictly hydrophobic (residue 35) and small, noncharged (residue 84) amino acids in all other characterized cystatins. Position 35 is thought to bind to the P3 site of the target protease (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar, 5Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar), so it is possible that this lysine substitution results in an especially high affinity for a cysteine protease with this preferred specificity. Because residues 81–85 in other cystatins usually form nonspecific hydrophobic interactions with the cognate protease (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar), it is likely that the contacts formed by this region may also differ from those observed previously. Supporting this, computer modeling has shown that Lys84 would interfere with binding of leukocystatin to papain in this region (see below).Leukocystatin contains a total of eight cysteines: the four that are conserved with other Class II cystatins, and four unique cysteines, two of which are in the leader region (Fig. 3). Conserved cysteine residues in the N-terminal portion are not seen in any other mature Class II cystatin molecule, although they do occur sporadically in other cystatin leader sequences. Two cysteine residues, in positions different from leukocystatin, also appear in the N-terminal region of each inhibitory kininogen domain, and a polymorphism in the cystatin D sequence introduces a cysteine in this area (18Freije J.P. Balbin M. Abrahamson M. Velasco G. Dalboge H. Grubb A. Lopez-Otin C. J. Biol. Chem. 1993; 268: 15737-15744Abstract Full Text PDF PubMed Google Scholar, 23Balbin M. Hall A. Grubb A. Mason R.W. Lopez-Otin C. Abrahamson M. J. Biol. Chem. 1994; 269: 23156-23162Abstract Full Text PDF PubMed Google Scholar). It is possible that the two additional leukocystatin cysteines in the putative mature protein form an intrachain disulfide and provide added stability. This, however, is not supported by the evidence. Participation of Cys63 in an intrachain disulfide could only occur if the leukocystatin structure is markedly different from chicken egg white cystatin or if the N terminus folds back because the structure of chicken egg white cystatin shows the amino acid corresponding to Cys63 at the end of an α-helix, 34 Å from the N terminus. This places it far from the other leukocystatin cysteines. Furthermore, nonreduced gels indicate that the long form, which contains Cys26, can dimerize (Fig. 8), whereas no evidence of dimerization exists for the short form, which contains Cys63 but not Cys26. Because only monomer is seen in reducing gels, this interaction is apparently mediated by an interchain disulfide, formed by Cys26 from two different molecules. This may be similar to the case of stefin B, a Class I cystatin, which has a cysteine at position 3 that is thought to mediate dimerization (24Turk V. Bode W. FEBS Lett. 1991; 285: 213-219Crossref PubMed Scopus (716) Google Scholar).The mouse leukocystatin gene contains four exons (Fig. 4), unlike most other members of the Class II cystatins, which have three (25Saitoh E. Isemura S. Crit. Rev. Oral Biol. Med. 1993; 4: 487-493Crossref PubMed Scopus (17) Google Scholar). Soyacystatin is the only known molecule containing one cystatin domain and having a gene encoding four exons. The additional exon in that case, however, lies in a unique C-terminal extension (26Misaka T. Kuroda M. Iwabuchi K. Abe K. Arai S. Eur. J. Biochem. 1996; 240: 609-614Crossref PubMed Scopus (93) Google Scholar). Because the amino acids encoded by the first two exons of leukocystatin are very different from other Class II cystatins, it is clear that the evolution of this region is very different from other family members. The C-terminal genomic organization, however, is similar to the other Class II cystatins, with the intron/exon boundaries being conserved. Furthermore, the N-terminal portion of leukocystatin is not similar to Class I cystatins: the Class I genomic organization is different, with the first intron lying at a position between the first and second leukocystatin introns and with the second lying between the second and third leukocystatin introns (27Pennacchio L.A. Myers R.M. Genome Res. 1996; 6: 1103-1109Crossref PubMed Scopus (33) Google Scholar, 28Sato N. Ishidoh K. Uchiyama Y. Kominami E. Gene. 1992; 114: 257-260Crossref PubMed Scopus (12) Google Scholar).Several forms of leukocystatin were produced in E. coli and were active cysteine protease inhibitors. Although some of these products require refolding, the FLAG-tagged material was soluble, similar to other Class II members expressed in E. coli, including those used for comparison in Table II (16Auerswald E.A. Genenger G. Assfalg-Machleidt I. Machleidt W. Engh R.A. Fritz H. Eur. J. Biochem. 1992; 209: 837-845Crossref PubMed Scopus (50) Google Scholar, 17Hall A. Hakansson K. Mason R.W. Grubb A. Abrahamson M. J. Biol. Chem. 1995; 270: 5115-5121Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Although the Class I cystatin A requires refolding following overexpression inE. coli, this was shown to have no adverse effect on activity (21Shibuya K. Kaji H. Itoh T. Ohyama Y. Tsujikami A. Tate S. Takeda A. Kumagai I. Hirao I. Miura K. Inagaki F. Samejima T. Biochemistry. 1995; 34: 12185-12192Crossref PubMed Scopus (22) Google Scholar, 22Shibuya K. Kaji H. Ohyama Y. Tate S. Kainosho M. Inagaki F. Samejima T. J. Biochem. 1995; 118: 635-642Crossref PubMed Scopus (13) Google Scholar). We further controlled for any effects that refolding may have on activity by examining denatured/renatured chicken egg white cystatin and found no difference in activity following this step.We determined the apparent K i values of leukocystatin with papain and cathepsin L. These are compared in TableII with published values for other Class II cystatins.K i values in the literature vary for the same cystatin-protease pair, probably due to the differing lengths of the N termini in various cystatin preparations; these residues are easily proteolyzed during isolation of native cystatins. In general, the affinity of cystatins for cathepsin B is much weaker than the binding to cathepsin L or papain, a trend that holds for the leukocystatins as well. In fact, no inhibition of cathepsin B was detected, although a reasonable apparent K i was found for chicken egg white cystatin. One possible explanation for the weaker association of leukocystatin to all examined proteases is the presence of lysine residues at positions 35 and 84, which replaces amino acids that are uncharged in every other known cystatin. Both residues are also known to be intimately involved in the binding process of these inhibitors to cysteine proteases (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar), and N-terminal truncation or substitution at either of these sites can lead to dramatic decreases in the ability of these cystatins to inhibit cysteine protease activity. For instance, some substitutions in the Gln81-Gly85 loop have been shown to be detrimental to binding, although position 84 was not itself modified in these studies (16Auerswald E.A. Genenger G. Assfalg-Machleidt I. Machleidt W. Engh R.A. Fritz H. Eur. J. Biochem. 1992; 209: 837-845Crossref PubMed Scopus (50) Google Scholar). Modeling of a lysine at position 84, based upon the known complexed structure of stefin B to papain (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar), indicated that this substitution would cause serious steric interactions at this interface in addition to penalties resulting from desolvation and burying the positive charge of the lysine side chain (data not shown). Furthermore, it has been shown that the residues preceding the conserved glycine 37 are important for binding because removal of these can reduce binding drastically (29Machleidt W. Thiele U. Laber B. Assfalg-Machleidt I. Esterl A. Wiegand G. Kos J. Turk V. Bode W. FEBS Lett. 1989; 243: 234-238Crossref PubMed Scopus (151) Google Scholar, 30Lindahl P. Abrahamson M. Bjork I. Biochem J. 1992; 281: 49-55Crossref PubMed Scopus (83) Google Scholar, 31Bjork I. Pol E. Raub-Segall E. Abrahamson M. Rowan A.D. Mort J.S. Biochem J. 1994; 299: 219-225Crossref PubMed Scopus (77) Google Scholar), in some cases largely due to slower association rates. For the leukocystatins, we have observed that the time to reach equilibrium is rather slow, taking several minutes. In our case, this may indicate a necessary conformational change in the protease and/or leukocystatin for binding to occur. Furthermore, Hall et al. (17Hall A. Hakansson K. Mason R.W. Grubb A. Abrahamson M. J. Biol. Chem. 1995; 270: 5115-5121Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) postulate that the residue at position 35 may be a primary determinant of protease specificity because these N-terminal residues associate with the protease binding sites (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar, 5Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar). Lindahl et al. (32Lindahl P. Ripoll D. Abrahamson M. Mort J.S. Storer A.C. Biochemistry. 1994; 33: 4384-4392Crossref PubMed Scopus (32) Google Scholar) have shown that an arginine substitution in cystatin C at the position equivalent to Pro36 can have a large impact on binding to cathepsin B or to papain and may even cause displacement of the N terminus from the protease (32Lindahl P. Ripoll D. Abrahamson M. Mort J.S. Storer A.C. Biochemistry. 1994; 33: 4384-4392Crossref PubMed Scopus (32) Google Scholar). Although that position is probably more critical to tight binding to the cognate protease than the residue at 35, it demonstrates that changes in the amino acids at these positions can greatly affect the ability of cystatins to inhibit cysteine proteases. It is therefore likely that the native binding partner of leukocystatin is unlike that of the examined proteases. It is possible that the target is some as yet unidentified lysosomal protease, or even a protease from a different family. For instance, the ubiquitin-hydrolase UCH-L3 has recently been shown by x-ray crystallography to have a papain-like fold, being particularly similar to cathepsin B in the active-site cleft (33Johnston S.C. Larsen C.N. Cook W.J. Wilkinson K.D. Hill C.P. EMBO J. 1997; 16: 3787-3796Crossref PubMed Scopus (213) Google Scholar), and so may very well be inhibited by cystatins, although no evidence of this is yet in the literature. Particularly intriguing is the fact that this isozyme is primarily found in hematopoietic cells (34Wilkinson K.D. Lee K.M. Deshpande S. Duerksen-Hughes P. Boss J.M. Pohl J. Science. 1989; 246: 670-673Crossref PubMed Scopus (755) Google Scholar, 35Wilkinson K.D. Deshpande S. Larsen C.N. Biochem. Soc. Trans. 1992; 20: 631-637Crossref PubMed Scopus (129) Google Scholar), and is specific for the RGG sequence of ubiquitin. Furthermore, a domain of kininogen has shown inhibitory activity against calpains (36Salvesen G. Parkes C. Abrahamson M. Grubb A. Barrett A.J. Biochem. J. 1986; 234: 429-434Crossref PubMed Scopus (176) Google Scholar), and legumain is inhibited by chicken egg white cystatin (37Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), demonstrating that other families of cysteine proteases can be inhibited by these sorts of structures.In support of a unique target for the leukocystatins, we found little difference in the binding abilities of long and short forms of cystatin with papain in the presence of 5 mm DTT. Based upon the results of experiments with chicken cystatin, in which N-terminally truncated forms were found to not be as efficient inhibitors as the full-length molecules (29Machleidt W. Thiele U. Laber B. Assfalg-Machleidt I. Esterl A. Wiegand G. Kos J. Turk V. Bode W. FEBS Lett. 1989; 243: 234-238Crossref PubMed Scopus (151) Google Scholar, 30Lindahl P. Abrahamson M. Bjork I. Biochem J. 1992; 281: 49-55Crossref PubMed Scopus (83) Google Scholar, 31Bjork I. Pol E. Raub-Segall E. Abrahamson M. Rowan A.D. Mort J.S. Biochem J. 1994; 299: 219-225Crossref PubMed Scopus (77) Google Scholar), we would expect to see dramatic differences in the abilities of these variants to inhibit this enzyme. One possible explanation is that the 8-amino acid N-terminal extension of the longer form impedes binding, although extensions have been shown to have little effect for other cystatins (38Hakansson K. Huh C. Grubb A. Karlsson S. Abrahamson M. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 1996; 114: 303-311Crossref PubMed Scopus (56) Google Scholar). Furthermore, the absence of an effect of the FLAG tag supports the idea that N-terminal extensions do not influence leukocystatin binding. The unique lysine at position 35 may, however, interfere with complex formation. There is also some evidence that the long forms dimerize, and this may interfere with binding. Under the assay conditions, however, a large proportion is likely to be monomeric, as evidenced by the titration shown in Fig. 8. That the interchain cysteine primarily mediates this dimerization is evidenced by the fact that activity increases for the long form with increasing DTT concentrations. If we were to assume that the dimeric form did not bind at all to the studied cysteine proteases (and that all of the inhibition resulted from the monomeric form) this would only result in changing the apparent K i by a factor of less than 2 (in favor of tighter binding), because the effective concentration would be changed by this amount.Although there was no effect on papain inhibition, there was a 10-fold increase in binding affinity to cathepsin L with the long form, showing that at least for this particular case, the N terminus contributes to binding. This supports the idea that the various portions of cystatins are differentially involved in association to individual proteases, even though the three-dimensional structures of these proteases are very similar. We would expect the native binding partner of leukocystatin to fully take advantage of the unique features in these sites.Leukocystatin was shown by cDNA library Southern blots to be expressed selectively in hematopoietic cells. Examination of a wide variety of immune cell types suggests that the highest levels are expressed in T-cells, monocytes, and dendritic cells. Clearly, a search for a specific target protease should focus on the effector functions of these immune cell types. Currently, we are developing other tagged versions of leukocystatin, additional antibody reagents, and a mouse gene knockout to probe in depth the biological role of this novel Class II cystatin.In conclusion, we have characterized a new Class II cystatin, termed leukocystatin, which has a novel sequence, including unique lysine residues at two important protease binding sites, and a distinct distribution in hematopoietic cells. Cysteine proteases play many very important roles in the immune system. For instance, the de-ubiquinating enzymes are cysteine proteases, whereas lysosomal proteases are involved in antigen presentation both through the degradation of proteins to antigenic peptides and by processing the invariant chain of class II major histocompatibility complexes (1Riese R.J. Wolf P.R. Bromme D. Natkin L.R. Villadangos J.A. Ploegh H.L. Chapman H.A. Immunity. 1996; 4: 357-366Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). However, the overexpression of these proteases can be detrimental to cells, as can their release into the extracellular space. Therefore, their activities in these cells are controlled by a variety of mechanisms, including the presence of macromolecular protease inhibitors. The cystatins make up a class of very tight, reversible, competitive inhibitors of the papain family of cysteine proteases. Cystatins have been divided into four classes based upon their sequences and properties. Class I, also called the stefins, are a group of intracellular proteins of approximately 100 residues that contain no disulfide bonds. Class II cystatins are secreted inhibitors of about 120 amino acids containing two disulfide bonds. Class III cystatins, known as the kininogens, contain three domains, each of which resembles Class II cystatins; two of these domains possess inhibitory activity. Finally, Class IV cystatins constitute a poorly understood group of glycoproteins with two nonfunctional cystatin domains. The amino acid sequences and genomic structures within each family are highly conserved. Cystatins are expressed throughout the body in a tissue-specific manner. Mutations in some cystatins or alterations in the balance of these with their cognate cysteine proteases have been implicated in several diseases (2Calkins C.C. Sloane B.F. Biol. Chem. Hoppe-Seyler. 1995; 376: 71-80PubMed Google Scholar, 3Henskens Y.M. Veerman E.C. Nieuw Amerongen A.V. Biol. Chem. Hoppe-Seyler. 1996; 377: 71-86Crossref PubMed Google Scholar). Many studies, involving changes of peptide sequence, have shown that three regions of the cystatin, which form a "wedge" that can associate with the active-site cleft, are all required for tight binding to the protease. These studies have been confirmed by the crystal structure of the cystatin B-papain complex (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar) and supported by other structural studies showing that chicken egg white cystatin, a Class II cystatin, has the same fold as the Class I cystatin B (5Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar, 6Dieckmann T. Mitschang L. Hofmann M. Kos J. Turk V. Auerswald E.A. Jaenicke R. Oschkinat H. J. Mol. Biol. 1993; 234: 1048-1059Crossref PubMed Scopus (81) Google Scholar). In this paper, we describe the characterization of a new Class II cystatin, leukocystatin, specifically expressed by hematopoietic cells. The unique features of the amino acid sequence suggest that the as yet unidentified target protease is not one of the commonly studied lysosomal cysteine proteases, although leukocystatin is an active inhibitor of these cathepsins. In addition, the unusual genomic structure of the mouse protein and the amino acid sequences of both the human and mouse inhibitors suggest that they are quite divergent from other Class II cystatins. DISCUSSIONWe have discovered a novel hematopoietic cell-specific Class II cystatin from an EST analysis of human dendritic cells. This protein, which we have called leukocystatin, has all the features of a Class II cystatin, but it has some notable characteristics. For example, leukocystatin contains lysine residues at two positions that are strictly hydrophobic (residue 35) and small, noncharged (residue 84) amino acids in all other characterized cystatins. Position 35 is thought to bind to the P3 site of the target protease (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar, 5Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar), so it is possible that this lysine substitution results in an especially high affinity for a cysteine protease with this preferred specificity. Because residues 81–85 in other cystatins usually form nonspecific hydrophobic interactions with the cognate protease (4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar), it is likely that the contacts formed by this region may also differ from those observed previously. Supporting this, computer modeling has shown that Lys84 would interfere with binding of leukocystatin to papain in this region (see below).Leukocystatin contains a total of eight cysteines: the four that are conserved with other Class II cystatins, and four unique cysteines, two of which are in the leader region (Fig. 3). Conserved cysteine residues in the N-terminal portion are not seen in any other mature Class II cystatin molecule, although they do occur sporadically in other cystatin leader sequences. Two cysteine residues, in positions different from leukocystatin, also appear in the N-terminal region of each inhibitory kininogen domain, and a polymorphism in the cystatin D sequence introduces a cysteine in this area (18Freije J.P. Balbin M. Abrahamson M. Velasco G. Dalboge H. Grubb A. Lopez-Otin C. J. Biol. Chem. 1993; 268: 15737-15744Abstract Full Text PDF PubMed Google Scholar, 23Balbin M. Hall A. Grubb A. Mason R.W. Lopez-Otin C. Abrahamson M. J. Biol. Chem. 1994; 269: 23156-23162Abstract Full Text PDF PubMed Google Scholar). It is possible that the two additional leukocystatin cysteines in the putative mature protein form an intrachain disulfide and provide added stability. This, however, is not supported by the evidence. Participation of Cys63 in an intrachain disulfide could only occur if the leukocystatin structure is markedly different from chicken egg white cystatin or if the N terminus folds back because the structure of chicken egg white cystatin shows the amino acid corresponding to Cys63 at the end of an α-helix, 34 Å from the N terminus. This places it far from the other leukocystatin cysteines. Furthermore, nonreduced gels indicate that the long form, which contains Cys26, can dimerize (Fig. 8), whereas no evidence of dimerization exists for the short form, which contains Cys63 but not Cys26. Because only monomer is seen in reducing gels, this interaction is apparently mediated by an interchain disulfide, formed by Cys26 from two different molecules. This may be similar to the case of stefin B, a Class I cystatin, which has a cysteine at position 3 that is thought to mediate dimerization (24Turk V. Bode W. FEBS Lett. 1991; 285: 213-219Crossref PubMed Scopus (716) Google Scholar).The mouse leukocystatin gene contains four exons (Fig. 4), unlike most other members of the Class II cystatins, which have three (25Saitoh E. Isemura S. Crit. Rev. Oral Biol. Med. 1993; 4: 487-493Crossref PubMed Scopus (17) Google Scholar). Soyacystatin is the only known molecule containing one cystatin domain and having a gene encoding four exons. The additional exon in that case, however, lies in a unique C-terminal extension (26Misaka T. Kuroda M. Iwabuchi K. Abe K. Arai S. Eur. J. Biochem. 1996; 240: 609-614Crossref PubMed Scopus (93) Google Scholar). Because the amino acids encoded by the first two exons of leukocystatin are very different from other Class II cystatins, it is clear that the evolution of this region is very different from other family members. The C-terminal genomic organization, however, is similar to the other Class II cystatins, with the intron/exon boundaries being conserved. Furthermore, the N-terminal portion of leukocystatin is not similar to Class I cystatins: the Class I genomic organization is different, with the first intron lying at a position between the first and second leukocystatin introns and with the second lying between the second and third leukocystatin in
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