INTRODUCTION

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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 (1). 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 (2, 3). 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 (4) 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 (5, 6).

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.

	MATERIALS AND METHODS

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General-- Antisera to the human protein (Josman Laboratories, Napa, CA), used for protein blotting, were produced in rabbits against the peptide GFPKTIKTNDPGVLQAAR, which was synthesized on a lysine matrix (Biosynthesis, Inc., Lewisville, TX). Protein blots were detected with the Enhanced Chemiluminescent Detection System (Amersham Pharmacia Biotech). Chicken egg white cystatin was from PanVera (Madison, WI). Automated DNA sequencing was performed with a DyeTerminator Cycle Sequencing Ready Reaction kit on an Applied Biosystems Prism 377 DNA sequencer (both from Perkin-Elmer). PCRs were performed on a Perkin-Elmer 9600 with a GeneAmp PCR kit (Perkin-Elmer) followed by purification with QIAquick Gel Extraction kits (Qiagen, Santa Clarita, CA). Oligonucleotides were synthesized using an Applied Biosystems 394 synthesizer (Perkin-Elmer).

cDNA Libraries-- cDNA libraries are listed on the Southern blots (see Figs. 5 and 6) and were made with the SuperscriptII system (Life Technologies Inc.) as detailed elsewhere (Refs. 7 and 8 and references therein). Details are also available directly from the authors. In cases where the derivation is not obvious from the name, conditions used are listed below. Mouse libraries: 2) Braf:ER transfectant NIH3T3 cell line, ethanol-treated; 3) Mel14 bright CD4+ cells from spleen, polarized for 7 days with IFN-¹ and anti-IL4, activated with anti-CD3 for 2, 6, or 16 h and pooled; 4) as for 3, except polarized with IL4 and anti-IFN-; 5) naive CD4+ cells from an ovalbumin-specific TcR transgenic mouse, polarized for 3 weeks with IL12 and anti-IL4, activated with PMA and ionomycin for 2, 6, or 24 h, and pooled; 6) as for 5, but polarized with IL4¹ and anti-IL12; 8) D1.1 TH1 cell clone, 3 weeks after last antigen stimulation; 9) D1.1 TH1 cell clone, concanavalin A-stimulated for 15 h; 10) CDC35 TH2 cell clone, 3 weeks after last antigen stimulation; 11) CDC35 TH2 cell clone, concanavalin A-stimulated 15 h; 12) Mel14+ naive T-cells from spleen; 13) as for 12, but polarized for 7 days with IFN-, IL12, and anti-IL4; 14) as for 13, but polarized with IL4 and anti-IFN-; 18) B-cells from LPS-induced total spleen; 19) splenic dendritic cells derived by metrizamide enrichment; 21) RAW 264.7 cells activated with LPS 4 h; 22) bone marrow-derived macrophages grown in granulocyte-macrophage CSF and macrophage CSF; 24) macrophage cell line treated with LPS and anti-IL10 for 0.5, 1, 3, 6, or 12 h and pooled; 25) as for 24, but treated with LPS and IL10. Rat libraries: 46) normal joint tissue, mock-injected; 47) pooled streptococcal cell wall antigen-induced arthritic joints. Human libraries: 50) natural killer cell clone derived from peripheral blood of a large granular lympocyte leukemia patient, treated with IL2; 51) 20 pooled natural killer cell clones activated with PMA and ionomycin for 6 h; 53) B-cell line JY activated with PMA and ionomycin for 1 or 6 h and pooled; 55) splenocytes activated with anti-CD40 and IL4; 57) pooled peripheral blood T-cell clones; 58) 6 pooled resting T-cell clones; 60) CD4+CD45RO T-cells polarized 27 days in anti-CD28, IL4, and anti-IFN-, activated with anti-CD3 and anti-CD28 for 4 h; 61) TH2 cell clone HY935 treated with anti-CD28 and anti-CD3 for 2, 7, or 12 h and pooled; 63) TH1 cell clone HY06 treated with specific antigenic peptide for 2, 6, or 12 h and pooled; 64) as for 63, but treated with anti-CD28 and anti-CD3; 66) TH0 cell clone Mot 72 treated with specific antigenic peptide for 2, 7, or 12 h and pooled; 67) as for 66, but treated with anti-CD28 and anti-CD3 for 3, 6, or 12 h and pooled; 69) peripheral blood mononuclear cells (monocytes, T-cells, natural killer cells, granulocytes, and B-cells) treated with anti-CD3 and PMA for 2, 6, or 12 h and pooled; 71) kidney epithelial carcinoma cell line CHA activated with PMA and ionomycin for 1 or 6 h and pooled; 72) lung fibroblast sarcoma line MRC5, treated as for 71; 73) hematopoietic precursor line TF1, treated as for 71; 74) malignant tumor leiomyosarcoma; 75) normal myometrium; 77) dendritic cells derived from elutriated blood monocytes after 5 days in granulocyte-macrophage CSF and IL4, activated with tumor necrosis factor , IL1, and monocyte supernatant for 4 or 16 h and pooled; 78) as for 77, but activated with LPS; 79) as for 77, but not activated; 80) as for 79, but from a different monocyte donor; 81) 95% CD1a+CD86+ dendritic cells derived from CD34+ stem cells after 12 days in granulocyte-macrophage CSF and tumor necrosis factor , FACS-sorted, activated with PMA and ionomycin for 1 or 6 h, and pooled; 82) as for 81, but with CD14+ cells; 83) as for 81, but with CD1a+ cells; 84) 70% CD1a+ dendritic cells derived from CD34+ stem cells after 12 days in granulocyte-macrophage CSF and tumor necrosis factor , activated with PMA and ionomycin for 6 h; 85) as for 84, but activated for 1 h; 86) as for 84, but not activated; 87) elutriated monocytes activated with LPS for 6 h; 88) elutriated monocytes activated with LPS for 1 h; 89) as for 87, but activated with LPS, IFN-, and IL10 for 4 or 16 h and pooled; 90) as for 89, but activated with LPS, IFN-, and anti-IL10; 91) as for 89, but from a different donor; 92) as for 90, but from a different donor; 93) U937 premonocytic cell line activated with PMA and ionomycin for 1 or 6 h and pooled; 94-105) from a 28-week-old fetus; 106) from a 28-week pregnancy; 107) from a 12-year-old patient.

Identification and Characterization of Human Leukocystatin cDNA-- An average of 375 base pairs of highly unambiguous sequence (EST) from individual clones from cDNA libraries 84 and 86 were determined. The sequences were analyzed for possible encoded function by BLASTN searches versus the public data bases, followed by BLASTP searches of the open reading frames (9). By this method, two leukocystatin ESTs (from a total of 1190) were identified. Both cDNAs were completely sequenced on each DNA strand and were found to be full-length.

Isolation and Characterization of Mouse Leukocystatin cDNA-- 160 pools of approximately 500 clones each from a mouse TH2 cDNA library (Ref. 10; Library 4) were amplified overnight to form sublibraries. Southern blots were performed on the sublibraries using a ³²P-labeled 321-base pair probe to the human sequence (see below), washing with cross-species conditions (2× SSC, 0.1% SDS at 65 °C). One of the 160 sublibraries showed a positive signal. A bacterial stock from this pool was plated out, and colony hybridization was conducted under the same conditions to yield several possible positive clones, one of which was selected and found to encode a full-length copy of mouse leukocystatin.

cDNA Library Southern Blots-- The method presented by Bolin et al. (7) was followed. Briefly, NotI/SalI digests of 5 µg of cDNA library released the cDNA inserts from the vector. Digestion reactions were run on 1% agarose gels, transferred to Nytran+ filters (Schleicher and Schuell), and cross-linked with a UV Stratalinker 1800 cross-linker (Stratagene, La Jolla, CA). For the human blot, a 321-base pair ³²P-labeled probe was synthesized with [³²P]dCTP (Amersham Pharmacia Biotech) using the Rediprime system (Amersham Pharmacia Biotech). Hybridization with 1.5 × 10⁶ cpm/ml was performed at 60 °C in ExpressHyb (CLONTECH, Palo Alto, CA) followed by washes in 0.5× SSC, 0.1% SDS. The 343-base pair ³²P-labeled mouse probe was made using a Prime-IT II kit (Stratagene) followed by purification on a Centrisep column (Princeton Separations, Adelphia, NJ). Hybridization was performed in 0.5 M sodium phosphate, pH 7.2, 7% SDS, 0.5 mM EDTA at 65 °C followed by washes in 0.1× SSC, 0.1% SDS. Intensities of the bands were quantitated with a Molecular Dynamics Personal Densitometer (Sunnyvale, CA) scan of the developed x-ray film (Kodak BioMax, Rochester, NY).

Genomic DNA Sequence-- A 129SV mouse genomic library (Stratagene) was screened with a 617-base pair ³²P-labeled probe (complementary to the mouse cDNA sequence) in QuikHyb (Stratagene) using conditions recommended by the manufacturer. Of approximately 10 million clones, 4 were identified as being positive. Through a series of PCRs using primers that hybridized to various portions of the leukocystatin sequence, one clone was shown to contain the entire gene. In order to fully sequence this gene, a series of PCRs (26 reactions total) were performed. The resulting overlapping fragments were sequenced in both directions, generating 12 kilobases of sequence consistent with the cDNA sequence.

Recombinant Protein Expression and Purification-- The PCR primers listed in Table I were used to amplify the leukocystatin sequence with appropriate restriction sites. These amplimers were subcloned into the BamHI/NotI or BamHI/EcoRI sites of pGEX-4T-1 (Amersham Pharmacia Biotech) or HindIII/EcoRI sites of pFLAG (IBI, Eastman Kodak). The coding regions of the constructs were completely sequenced, and DNA preparations using QIA filter plasmid maxi kits (Qiagen) were made for transformation into the Escherichia coli strains used for protein expression.

F, thi-1

Refolding of Chicken Egg White Cystatin-- 2 mg of chicken egg white cystatin was concentrated to 100 µl and then incubated in 1 ml of 8 M guanidinium chloride in 50 mM Tris-HCl, pH 8.25, 10 mM DTT at room temperature for 2 h. The unfolded protein was diluted into 50 mM Tris-HCl, pH 8.25, 2.5 mM reduced glutathione, 1.0 mM oxidized glutathione and incubated for 12 h at 4 °C. Following dialysis into 50 mM Tris-HCl, pH 8, the refolded material was purified on a Poros reverse phase column, eluting with a 2%  80% acetonitrile gradient containing 0.1% trifluoroacetic acid. The fractions containing active inhibitor were quantitated as for leukocystatin.

Inhibition Studies-- The method described by Abrahamson (11) was used, with some alterations. Papain activity (Sigma; final concentration, 290 pM) was fluorimetrically monitored (SPEX Fluorolog, Instruments SA, Edison, NJ) with 5-25 µM carbobenzoxy-L-phenylalanyl-L-arginine-7-amino-4-methylcoumarin (Bachem, King of Prussia, PA) in 100 mM sodium phosphate, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, 1% N,N-dimethylformamide, 0.01% Brij-35, pH 6.7, at room temperature (25 °C). The inhibition of human liver cathepsin B (290 pM; Calbiochem, San Diego, CA) and human liver cathepsin L (170 pM; Calbiochem) were determined in 100 mM NaOAc, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, 1% N,N-dimethylformamide, 0.01% Brij-35, pH 5.5. The substrates were carbobenzoxy-L-argininyl-L-arginine-7-amino-4-methylcoumarin (Bachem) and carbobenzoxy-L-phenylalanyl-L-arginine-7-amino-4-methylcoumarin for the two cathepsins, respectively. Uninhibited rates, v_o, were measured, and then inhibitor was added, ensuring that in all studies, at least 1 eq of inhibitor was present. The reaction was allowed to re-equilibrate for 25 min (a time course showed that equilibrium was achieved at this point for this range of inhibitor concentrations), and the inhibited rate, v_i, was determined. Apparent K_i values were determined from the equation [I]_t/(1  v_i/v_o) = E_t + (K_iv_o/v_i){1 + ([S]/K_m)} (12, 13) using Kaleidagraph software (Synergy Software, Reading, PA). All correlation coefficients were greater than 0.9.

	RESULTS

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Mouse and Human Leukocystatin cDNA Cloning and Sequence-- Almost 1200 ESTs were determined from a cDNA library of resting and activated human dendritic cells. Of these, 67% corresponded to sequences in the GenBank^TM data base with known function (as of May 1996) or to repeat elements. Included among these were the known cystatins A (three copies), B (one copy), and C (four copies). Many of the remaining sequences were identical to sequences in public EST data bases. Of the unknown sequences, two corresponded to the same 867-base pair cDNA and appeared to encode a protein related to known cystatins. Further sequence analysis of these clones confirmed this notion, revealing a full-length open reading frame corresponding to the protein we now designate leukocystatin. Two possible in-frame start codons, at nucleotide positions 66 to 63 and 1-3, are present within the N-terminal sequence (Fig. 1). We believe that the second is the one actually used in protein translation because this is the one that is conserved with the mouse sequence (see below) and is most in agreement with alignment to other cystatins. In addition, the sequence 66 to 1 would not code for a signal sequence, which would be inconsistent with the other features of this sequence, indicating that leukocystatin is a Class II cystatin (see below).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   cDNA sequence of human leukocystatin. The coding sequence is in boldface, with the corresponding protein sequence underneath. The upstream in-frame ATG discussed in the text is underlined. This sequence has been deposited in the GenBankTM under accession no. AF031824.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   cDNA sequence of mouse leukocystatin. The coding sequence is in boldface, with the corresponding protein sequence underneath. The last base of each exon is starred. The position of the polyadenylation signal is underlined. This has been deposited in the GenBankTM data base under accession no. AF031825.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Sequence alignment of leukocystatin protein sequences with chicken egg white cystatin. Numbering of preprotein amino acid sequences (as deduced from cDNA sequences) is according to the human sequence and begins at the putative initiating methionine. Dashes indicate gaps inserted for an optimal alignment. Asterisks indicate the end of the protein sequence, as determined by Opal codons in the cDNA sequence. Arrows indicate intron/exon junctions determined for the mouse leukocystatin sequence. The conserved cysteine residues defining Class II cystatins are shaded, and the residues identified by others as being critical to protease inhibition are shaded and boxed. The additional four cysteine residues unique to leukocystatin are boxed. The end of the predicted signal sequence is marked by . The sequence of chicken egg white cystatin (GenBankTM accession no. J05077) is given for comparison.

Genomic Structure-- A mouse genomic sequence corresponding to leukocystatin was found, and approximately 12 kilobases was sequenced. The mouse leukocystatin gene (deposited in the GenBank^TM data base under accession no. AF031826) contains four exons (Fig. 4). The N-terminal intron represents an additional break in the sequence relative to other Class II cystatin genes, splitting the codon for Asp²⁴. This first intron is particularly large, about 5 kilobases. The sites of the two C-terminal exon-intron junctions are relatively conserved in comparison with other known Class II and Class III junctions from animals. The second intron, lying between the codons for Gln⁸¹ and Val⁸², is approximately 1.8 kilobases, whereas the third is 0.6 kilobases and is located between the codons for Arg¹²⁰ and Thr¹²¹. The sequences at all the junctions are in agreement with GT/AG consensus sequences.

View larger version (2K): [in this window] [in a new window]   Fig. 4.   Schematic of genomic structure. The exons are indicated by the dark boxes, and introns are indicated by the connecting lines. The number of base pairs in each region is indicated underneath. The positions of the start and stop codons are indicated above the diagram. The determined DNA sequence has been deposited in the GenBankTM data base under accession no. AF031826.

mRNA Tissue Distribution-- Using cDNA library Southern blots (7), we determined the tissue distribution of mouse and human leukocystatin mRNAs (Figs. 5 and 6). This technology utilizes high quality representational cDNA libraries in place of the more typical Northern blots and is especially useful for examining mRNA tissue distributions in cases where these mRNAs are particularly hard to attain. We have confirmed many of these results with semiquantitative PCR (8, data not shown). In an analysis that addressed 61 human and 47 rodent cell types and/or activation conditions, human leukocystatin mRNA was found mainly in resting T-cells, premonocytic cells, activated dendritic cells derived from stem cells, and some natural killer cell clones. Interestingly, however, human leukocystatin was not seen in dendritic cells derived from monocytes, only in those from stem cells. Many differences between these two cell-types have been observed by others, although it is not clear what these differences mean functionally (15). The mouse leukocystatin mRNA, in contrast, is found primarily in differentiated T-cells, although there seems to be little difference in the levels between TH1 and TH2 cells; however, little is found in naive and pre-T-cells. A moderate amount is also found in monocytes, whereas B-cells, dendritic cells, and some macrophage libraries show small amounts of cDNA corresponding to this protein. The small amounts seen in lymph nodes, thymus, and spleen probably result from resident lymphocytes. The absence of readily-detectable mouse leukocystatin mRNA in splenic and bone marrow mouse dendritic cells may reflect their lineage.

View larger version (73K): [in this window] [in a new window]   Fig. 5.   Tissue distribution analysis of mouse leukocystatin mRNA. Each lane contains 5 µg of a digested cDNA library. All cDNA libraries and blot procedures are described under "Materials and Methods." The major band corresponds to 0.9 kilobases, as determined by molecular size markers. The minor band corresponds to background hybridization to the pSPORT vector. The audioradiograph was scanned, and levels of density of the 0.9-kilobase band relative to the maximal signal were plotted. The normal and arthritic joint tissues are from rat.

View larger version (60K): [in this window] [in a new window]   Fig. 6.   Tissue distribution analysis of human leukocystatin mRNA. Each lane contains 5 µg of a digested cDNA library. All cDNA libraries and blot procedures are described under "Materials and Methods." The major band corresponds to 0.9 kilobases, as determined by molecular size markers. The minor band corresponds to background hybridization to the pSPORT vector. The audioradiograph was scanned, and levels of density of the 0.9-kilobase band relative to the maximal signal were plotted.

Protein Production-- Proteins of two types were made in order to study the role of the N terminus in binding: the short form beginning with the conserved glycine at position 37, and the long form commencing with Gly¹⁹. Gly¹⁹ is predicted to be the last amino acid of the signal sequence, and so this version should represent the complete mature protein. Recombinant leukocystatin was produced in E. coli as either an N-terminal GST or FLAG fusion. Similar methods have been used previously to express other family members (16-20). The FLAG-tagged material was isolated as soluble material from the periplasm; the GST fusion, however, was primarily recovered as inclusion bodies which had to be refolded. Although the Class I cystatin A requires refolding after expression in E. coli (21, 22), no other Class II cystatin is insoluble when expressed in E. coli, even when expressed as the GST fusion (20). The mature protein was isolated following thrombin cleavage of the GST moiety or enterokinase cleavage of the FLAG tag. SDS-polyacrylamide gel electrophoresis showed the expected molecular weights and that the proteins were reasonably pure (Fig. 7). Nonreduced gels, however, suggest that the long form may exist primarily as a dimer (Fig. 8): as DTT concentrations are varied from 0 to 8 mM, this protein product exists as a dimer and as a monomer, respectively, as determined by apparent molecular weights. Amino acid sequencing and detection with a leukocystatin-specific antibody confirmed that the correct proteins were isolated. Because freeze-drying the purified protein resulted in material that could not be resolubilized, the protein was stored at pH 4.0 at 4 °C.

View larger version (74K): [in this window] [in a new window]   Fig. 7.   Silver-stained SDS-polyacrylamide gel electrophoresis of recombinant leukocystatins. Each lane contains approximately 50 ng of protein. Lane A, molecular weight markers. Lane B, lysozyme. Lane C, chicken egg white cystatin. Lane D, human short leukocystatin. Lane E, human long leukocystatin. Lane F, FLAG-human short leukocystatin. Lane G, FLAG-human long leukocystatin. Lane H, mouse short leukocystatin.

View larger version (71K): [in this window] [in a new window]   Fig. 8.   Nonreducing SDS-polyacrylamide gel electrophoresis of FLAG-human long leukocystatin treated with varying amounts of DTT. 50 ng of FLAG-human long leukocystatin in the presence of 8.0 (lane B), 4.0 (lane C), 1.6 (lane D), 0.8 (lane E), 0.4 (lane F), or 0 mM (lane G), DTT as indicated. Lane A contains molecular mass markers.

Inhibition of Cysteine Proteases-- We studied the inhibition of three cysteine proteases (papain, cathepsin B, and cathepsin L) by normal methods (11). All studies were carried out near the optimal pHs of the proteases, and also in the presence of 5 mM DTT, which may partially denature the cystatins but was necessary to maintain maximal activity of the cysteine proteases. Using this method, we confirmed that chicken egg white cystatin binds tightly to cysteine proteases: an apparent inhibition constant of 90 pM was obtained versus cathepsin B, whereas binding was too tight to papain to quantitate. Identical results were obtained following denaturing and renaturing of the chicken egg white cystatin (data not shown). These results are consistent with published values (Table II). We found that binding of leukocystatin to the cysteine proteases studied is slow and is also weaker than other Class II cystatins (Table II). In fact, we could detect no inhibition of cathepsin B activity with leukocystatin (lower limit of detection, K_d approximately 200 nM), although subnanomolar inhibition constants were found versus papain and cathepsin L. Whereas the different N-terminal forms of leukocystatin had little effect on the inhibition of papain, a 10-fold increase in affinity for cathepsin L was seen with the leukocystatin long form relative to the short form. Finally, although an affinity could not be determined quantitatively, we found that a cysteine-linked dimer of the leukocystatin long form was not as effective as a papain inhibitor as the reduced form. So, although leukocystatin is a functional Class II inhibitor, its unique amino acid sequence appears to interfere with binding to the commonly assayed cysteine proteases.

View this table: [in this window] [in a new window]   Table II Apparent inhibition constants Inhibition constants (expressed in nM units) were determined by the procedure of Abrahamson, modified as detailed under "Materials and Methods" using the equation [I]t/(1  vi/vo) = Et + (Kivo/vi) {1 + ([S]/Km)} with carbobenzoxy-L-phenylalanyl-L-arginine-7-amino-4-methylcoumarin (papain, catL) or carbobenzoxy-L-argininyl-L-arginine-7-amino-4-methylcoumarin (catB) as substrate at pH 6.7 (papain) or pH 5.5 (catB, catL). Published values corresponding to chicken egg white cystatin and cystatin C inhibition are included for reference. The long form corresponds to amino acids Gly19-His/Gln146; the short form corresponds to Gly37-His/Gln146; and FLAG forms include the N-terminal DYKDDDDKL sequence. The human long and short forms prepared by enterokinase cleavage of the FLAG-tagged material had activities identical to that prepared from the GST fusion. Chicken cystatin denatured and renatured as described under "Materials and Methods" had activity identical to the native protein.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We 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 (4, 5), 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 (4), 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 (18, 23). 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 -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 (24).

The mouse leukocystatin gene contains four exons (Fig. 4), unlike most other members of the Class II cystatins, which have three (25). 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 (26). 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 (27, 28).

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 (16, 17). Although the Class I cystatin A requires refolding following overexpression in E. coli, this was shown to have no adverse effect on activity (21, 22). 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 Table II 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 (4), 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 (16). Modeling of a lysine at position 84, based upon the known complexed structure of stefin B to papain (4), 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 (29-31), 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. (17) 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 (4, 5). Lindahl et al. (32) 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 (32). 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 (33), 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 (34, 35), and is specific for the RGG sequence of ubiquitin. Furthermore, a domain of kininogen has shown inhibitory activity against calpains (36), and legumain is inhibited by chicken egg white cystatin (37), 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 (29-31), 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 (38). 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.