Human HtrA, an Evolutionarily Conserved Serine Protease Identified as a Differentially Expressed Gene Product in Osteoarthritic Cartilage*

The human homologue of the Escherichia coli htr A gene product was identified by the differential display analysis of transcripts expressed in osteoarthritic cartilage. This transcript was identified previously as being repressed in SV40-transformed fibroblasts (Zumbrunn, J., and Trueb, B. (1996) FEBS Lett. 398, 187–192). Levels of HtrA mRNA were elevated ; 7-fold in cartilage from individuals with osteoarthritis compared with nonarthritic controls. Differential expression of human HtrA protein was confirmed by an immunoblot analysis of cartilage extracts. Human HtrA protein expressed in heterologous systems was secreted and exhibited endoproteolytic activity, including autocatalytic cleavage. Conversion by mutagenesis of the putative active site serine 328 to alanine eliminated the enzymatic activity. Serine 328 was also found to be required for the formation of a stable complex with a 1 -antitrypsin. We have determined that the HtrA gene is highly conserved among mammalian species: the amino acid sequences encoded by HtrA cDNA clones from cow, rabbit, and guinea pig are 98% identical to human. In E. coli , a functional htr A gene product is required for cell sur-vival after heat shock or oxidative stress; its role appears to be the degradation of denatured proteins. We propose that mammalian HtrA, with the addition of a new functionality during evolution, i.e. a mac25 homology domain, plays an important role in cell growth regulation. Cloning Mammalian Homologues of HtrA— Cow HtrA was isolated from a lung cDNA phage library (CLONTECH) by hybridization screening using the human cDNA as a probe. HtrA cDNA fragments were isolated by PCR from rabbit and guinea pig liver cDNA using primers designed from the human coding sequence corresponding to regions of maximum amino acid sequence identity with E. coli htr A. The DNA sequences of the derived clones were determined as described above. Additional PCR primers were then designed and used for 5 9 and 3 9 RACE to obtain extended cDNA clones.

Osteoarthritis (OA), 1 the most prevalent form of degenerative joint disease, involves chondrocyte loss and the breakdown of extracellular matrix components, leading to cartilage degeneration and the eventual deterioration of joint function (1). From a therapeutic perspective, it is important to understand the molecular events triggering the onset of OA and the biochemical pathways responsible for the disease progression that appear to be influenced by a complexity of environmental and genetic factors (2)(3)(4). Chondrocytes, the exclusive cell type in cartilage, maintain the integrity of the collagen/proteoglycan network by responding to a variety of stresses, including the normal mechanical load as well as abnormal trauma and injury (5). The cellular response to stress stimuli occurs through the regulation of a myriad of signal transduction pathways, leading to alterations in gene expression.
Analysis of differential gene expression using various molecular biological techniques has been increasingly applied to investigate complex biological phenomena (6). As an approach to study the molecular pathobiology of OA, we have used a mRNA differential display (7) to screen for differences in gene expression between osteoarthritic and nonarthritic cartilage. A major advantage of this methodology is the capability to amplify minute amounts of transcripts and rapidly determine their nucleotide sequences. We have identified over 120 transcripts that appear to be differentially expressed in OA cartilage, only 29 of which correspond to known proteins. 2 In this report, we describe the identification and characterization of one transcript (provisionally named ORF480) and its translation product, both of which are expressed at elevated levels in OA cartilage. The nucleotide sequence of ORF480 is identical to a recently described transformation-sensitive cDNA isolated from human fibroblasts (8). ORF480 codes for a protein with distinct domains of homology to human mac25 (9) and to a bacterial serine protease (HtrA) that is critical for the cellular response to thermal and oxidative stress (10,11). This report provides the first evidence that the ORF480-encoded protein (human HtrA) exhibits autocatalytic cleavage as well as endoproteolytic activity against an exogenous substrate, ␤-casein. When incubated in the presence of serum, HtrA protein binds to and forms a stable complex with ␣ 1 -antitrypsin. In addition, we have determined that the sequence of the HtrArelated domain of ORF480 is highly conserved among mammalian species. These findings open new avenues of investigation toward an understanding of the biological function(s) of mammalian HtrA.

EXPERIMENTAL PROCEDURES
Materials-Total RNA from OA and nonarthritic human cartilage was isolated according to published methods (12) and supplied to our laboratories by Drs. I. Patel and A. Amin (Hospital for Joint Diseases, New York University Medical School, New York, NY). Independent biochemical analyses of the isolated cartilage 3 as well as the differential mRNA expression of type II and type III collagens (see "Results") were consistent with the indicated pathological state of the samples used in this study.
mRNA Differential Display and RT-PCR Product Identification-First-strand cDNA was synthesized from 0.2 g of total RNA with each of the three anchored oligodeoxythymidylic acid primers from Gen-Hunter Corp. The reaction (20 l) was carried out at 37°C for 60 min. For PCR amplification, 1 l of the cDNA served as the template in a 10-l reaction mix containing 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl 2 , * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM were subjected to 40 cycles of amplification under the  following conditions: denaturing at 94°C for 30 s, annealing at 40°C for  2 min, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. The resulting PCR products were resolved on a denaturing polyacrylamide gel and visualized by autoradiography of the dried gel. PCR products of interest were excised from the gel, and the DNA was eluted and reamplified by PCR using the same primers and conditions described above, excluding the labeled nucleotide. PCR products were resolved on a 1.5% agarose gel, excised, and ligated into cloning vector PCR II 2.1 (TA cloning kit; Invitrogen). Clones of the PCR-generated fragments were obtained by transformation of Escherichia coli strain DH5␣ (Life Technologies, Inc.). DNA sequences were determined for at least three independent clones of each fragment using Dye Terminator Cycle Sequencing on an ABI PRISM 377 DNA sequencing system (Perkin-Elmer).
Semiquantitative RT-PCR-First-strand cDNA was synthesized from total RNA isolated from OA and nonarthritic cartilage. 200 ng of total RNA and 10 pmol of primer T 30 VN (where V ϭ A, C, and G and N ϭ A, C, G, and T) were mixed in a 6-l volume, heated to 72°C for 3 min, and quenched on ice for 3 min. Reverse transcription reactions (10 l) were prepared with final concentrations of 50 mM Tris-HCl (pH 8.3), 6 mM MgCl 2 , 75 mM KCl, 1 mM deoxynucleotide triphosphates, and 10 units of Moloney murine leukemia virus reverse transcriptase. This mixture was incubated at 42°C for 1.5 h and at 94°C for 5 min and quenched on ice. Serial dilutions of cDNA from different individuals were used for PCR amplification with a primer set for actin (forward primer, GGAGTCCTGTGGCATCCACGAAACTAC; reverse primer, CACATCTGCTGGAAGGTGGACAGCG) in a 25-l reaction volume with 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.001% gelatin, 20 M deoxynucleotide triphosphates, and 1.25 units of Ampli-Taq Gold Polymerase (Perkin Elmer). PCR was performed for 32 cycles (94°C for 30 s, 63°C for 30 s, 72°C for 2 min, and a final incubation at 72°C for 7 min). Nine l of the reaction mix were run on a 10% polyacrylamide gel, stained with SYBR TM Green I (Molecular Probes), and quantified using fluorescence imaging and ImageQuant software (Molecular Dynamics). Concentrations were selected to ensure that the reaction was within the log phase of amplification. These conditions were used for PCR reactions to quantify the relative levels of expression for 49A50/58A5 (ORF480), Hsp60, type II collagen, and type III collagen. Levels of expression were normalized to the levels of actin for each sample. A negative control reaction with no template was carried out for each primer set to verify the absence of contamination.
5Ј RACE Cloning of ORF480 from OA Cartilage-Separate firststrand cDNA syntheses were carried out with 500 ng of total RNA isolated from four OA cartilage samples. Aliquots were combined for the synthesis of double-stranded cDNA with anchors ligated at both ends according to the Marathon TM cDNA system (CLONTECH). An antisense primer derived from the sequence of clones 58A5/49A50 (TGTG-CATTGACCTTTGGGTGCTGAC) and an anchor-specific primer were used for 5Ј RACE stepdown PCR. The reaction mix contained 15 mM KOAc, 3.5 mM MgOAc, 75 g/ml bovine serum albumin, 0.2 mM deoxynucleotide triphosphates, and KlenTaq-1 DNA polymerase mix (CLONTECH). The reaction times and temperatures were as follows: 94°C for 5 min; five cycles of 94°C for 30 s and 72°C for 2 min; five cycles of 94°C for 30 s and 70°C for 2 min; 25 cycles of 94°C for 30 s and 68°C for 2 min, and a final incubation at 72°C for 7 min. Reaction products were run on a 1.4% agarose gel. DNA fragments between 1.5-3 kb and between 1-1.5 kb were isolated from the gel and cloned using the TA cloning kit (Invitrogen). Colonies were screened by PCR with two ORF480-specific primers. The clones with the longest inserts were identified by PCR using T7 and M13 (reverse) primers. Plasmid DNA from clones was prepared and sequenced as described above.
Isolation of ORF480 from MRHF Human Fibroblasts-The cDNA for ORF480 was generated by PCR using cDNA derived from MRHF fibroblast RNA (human foreskin fibroblasts; BioWhittaker) as the template. Initially, PCR primers AAACGGATCCACCATGCAGATCCCGCGC-GCC and AAACGAATTCCTATGGGTCAATTTCTTCGGG corresponding to the 5Ј and 3Ј ends of the coding region of GenBank accession number D87258 were used. PCR was performed with Pfu polymerase for 25 cycles (94°C for 1 min, 58°C for 1 min, and 72°C for 3 min). However, due to the high GC content within a region 5Ј of the unique HindIII site, PCR with this pair of primers generated a cDNA fragment with an internal deletion of 380 bp within the GC-rich region. The sequence of the DNA fragment 3Ј of the unique HindIII site was correct. Therefore, primers AAACGGATCCAGAGTCGCCATGCAGATC and TTGTCACGATCAGTCCATCT were used to generate a DNA fragment 5Ј of the unique HindIII site. PCR conditions were modified to include 10% dimethyl sulfoxide, which allowed the generation of the correctly amplified fragment. The resulting two DNA fragments were joined together at the HindIII site and subcloned into the BamHI/EcoRI sites of pcDNA3 (Invitrogen). The final cDNA fragment of ORF480 corresponds to nucleotides 38 -1491 of D87258.
Immunoblot Analysis of HtrA-related Protein in Cartilage Extracts-Frozen milled human cartilage (50 mg) was extracted with 100 l of 50 mM Tris-HCl buffer containing 1 M NaCl and a mixture of protease inhibitors (Boehringer Mannheim) at 4°C. Samples containing approximately 50 g of total protein were prepared for SDS gel electrophoresis and immunoblot analysis with anti-HtrA antiserum using the ECL detection system (Amersham). Rabbit antiserum was generated against the human HtrA domain (residues 191-480) expressed in E. coli. 4 This antiserum contains some immunoreactivity to albumin.
Generation of S328A Mutation in ORF480 -The S328A mutation was generated using the QuikChange site-directed mutagenesis kit (Stratagene). To avoid difficulty in primer extension with Pfu polymerase through the GC-rich region located 5Ј of the unique HindIII site of ORF480 cDNA, we cloned a DNA fragment corresponding to amino acid residue 161 to the end of the coding region into pcDNA3 and used it as a template. Primers used for the mutagenesis reaction were CATCAAC-TATGGAAACGCGGGAGGCCCGTTAG and CTAACGGGCCTC-CCGCGTTTCCATAGTTGATG. After confirming by sequence analysis that the correct mutation had been generated, the full-length ORF480 was reassembled into pcDNA3. The resulting plasmid was designated pcDNA3-ORF480-S328A.
In Vitro Translation of ORF480 -The translation product of ORF480 was synthesized in vitro using pcDNA3-ORF480 and pcDNA3-ORF480-S328A as templates in the TNT T7 Coupled Reticulocyte Lysate System (Promega), incorporating [ 35 S]methionine. The reaction products were separated on a 10% SDS-polyacrylamide gel. At the end of the run, the gel was dried under a vacuum at 80°C and analyzed on a PhosphorImager TM (Molecular Dynamics).
Heterologous Expression of ORF480 -Human embryonic kidney cells, 293 (American Type Culture Collection), were maintained in Minimal Essential Medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and 1ϫ antibiotic-antimycotic solution (Life Technologies, Inc.) at 37°C in a humidified CO 2 incubator. Cells were stably transfected with the pcDNA3-ORF480 expression vector using the ProFection Mammalian Transfection System (Promega). Clones were then selected by incubation with G418 (Life Technologies, Inc.) at a concentration of 400 g/ml and incubated in serum-free medium for expression of ORF480. Sf9 insect cells were maintained as suspension cultures at 28°C in Sf-900II SF medium. Recombinant baculovirus stocks carrying the ORF480 cDNA were generated utilizing the pFASTBAC1/ORF480 donor plasmid and the BAC-TO-BAC Baculovirus Expression System (Life Technologies, Inc.). Optimal infection conditions were determined by varying the multiplicity of infection and time course. Expression of the secreted ORF480 protein in both heterologous systems was confirmed by the immunoblot blot analysis of culture supernatants using anti-HtrA antiserum as described above. For immunoblot analyses to discern the presence of high molecular weight complexes of HtrA protein (Fig. 6), we used anti-HtrA antiserum that had been depleted of antialbumin cross-reactivity by affinity chromatography. 5 To monitor the protease activity of ORF480-encoded protein, 75 l of culture medium were incubated with 50 g of ␤-casein (Sigma) in 50 mM Tris-HCl (pH 7.5) for 1 h at 37°C, and the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis using either standard 12% or 4 -20% gradient gels (Bio-Rad Laboratories).
Genomic Blot Hybridization-A ZOO-BLOT (CLONTECH) membrane filter containing EcoRI-digested genomic DNA from various species was prehybridized for 30 min at 65°C and then hybridized with a random-primed 32 P-labeled BamHI-EcoRI (ϳ900 bp; HtrA-related domain) fragment of ORF480 in rapid hybridization buffer (Amersham) at 65°C for 90 min. The hybridized filter was washed with 2ϫ SSC (sodium chloride/sodium citrate)/0.1% SDS for 20 min at room temperature (twice for 10 min at 65°C and washed once with 0.1ϫ SSC/0.1% SDS at 65°C for 10 min. The results were visualized using a Phosphor-Imager TM (Molecular Dynamics).
Cloning Mammalian Homologues of HtrA-Cow HtrA was isolated from a lung cDNA phage library (CLONTECH) by hybridization screening using the human cDNA as a probe. HtrA cDNA fragments were isolated by PCR from rabbit and guinea pig liver cDNA using primers designed from the human coding sequence corresponding to regions of maximum amino acid sequence identity with E. coli htrA. The DNA sequences of the derived clones were determined as described above. Additional PCR primers were then designed and used for 5Ј and 3Ј RACE to obtain extended cDNA clones.

Identification, Cloning, and Sequence Analysis of ORF480
cDNA-In the course of screening for differences in gene expression between osteoarthritic and nonarthritic cartilage by mRNA differential display, two PCR fragments, designated 49A50 and 58A5, were identified using different arbitrary primers (Fig. 1). Sequence analysis of 49A50 and 58A5 demonstrated that the two cloned PCR products correspond to the 3Ј end of the same mRNA (data not shown). Using the 5Ј RACE technique (13), a ϳ1.4-kb PCR fragment corresponding to 49A50/58A5 was isolated and cloned from OA cartilage-derived cDNA. The DNA sequences of two independent clones derived from RACE products C05 and C13 revealed overlapping open reading frames of 337 and 328 codons, respectively. Because no ATG initiation codon was evident in the 5Ј sequences of clones C05 and C13, and Northern blot data indicated a mRNA size of over 2 kb (data not shown), it was evident that these cartilagederived cDNA clones did not represent the entire transcript. At this point in the study, a BLAST (14) search of the GenBank and EST databases identified two 3Ј EST sequences (GenBank accession numbers W47107 and W67176) and a 2036-bp cDNA entry (GenBank accession number D87258). The translated sequence of GenBank entry D87258 contains an open reading frame of 480 amino acids (ORF480). The DNA sequence of clone C05 was identical to bases 477-1646 of D87258, except for a 1-bp difference (a probable PCR-generated error). Thus, the cDNA clones isolated from OA cartilage and the D87258 sequence cloned from osteoblasts represent the same gene product. RT-PCR analysis indicated that the mRNA for ORF480 is expressed in human placenta and in normal human dermal fibroblasts (data not shown). Overlapping PCR-generated fragments corresponding to the entire ORF480 were isolated from cDNA derived from fibroblast RNA (see "Experimental Procedures"). The DNA sequence of the fibroblast-derived ORF480 cDNA was determined to be identical to that of GenBank entry D87258. An alignment of the cDNA clones described in this report in relation to those identified from searching the databases is illustrated in Fig. 2. Also shown in Fig. 2 are the deduced protein domains within ORF480 as described below.
During the course of the work described here, Zumbrunn and Trueb (8)  Our analysis of the ORF480 sequence, although in general agreement, differed somewhat from that of Zumbrunn and Trueb due to differences in searching methods and the scope of the databases used. Using BLASTP to search the GenBank protein sequence data base, we found human mac25, a presumed member of the IGF-binding protein family (9), to have the highest degree of homology to the amino-terminal domain of ORF480 (Fig. 3A). For instance, the p value for the mac25 alignment with ORF480 residues 107-158 is 1.4 ϫ 10 Ϫ23 , compared with p ϭ 3.8 ϫ 10 Ϫ8 for insulin-related growth factorbinding protein 3. Interestingly, unlike IGF-binding proteins, the sequence of mac25 within the region homologous to ORF480 contains a conserved Kazal-type serine protease inhibitor motif (Fig. 3). Moreover, as pointed out by Kato et al. (16), mac25 is more closely related to follistatin than to insulinrelated growth factor-binding protein, and we find that the sequence of follistatin also contains the Kazal-type inhibitor motif (Fig. 3).
Differential Expression of ORF480 mRNA and HtrA Protein in OA Cartilage-To verify the initial differential display results and quantify the relative difference in the mRNA levels of human HtrA (ORF480) in OA cartilage compared with nonarthritic cartilage, oligonucleotide primer pairs specific for a 3Ј segment of ORF480 cDNA were used for semiquantitative RT-PCR (Fig. 4A) The results of this analysis, using expression levels of ␤-actin and Hsp60 for comparison, indicate that HtrA mRNA is present at levels that are ϳ7-fold higher in OA cartilage than in nonarthritic controls. Also included in Fig. 4A are data for both type II (COL2A) and type III (COL3A) collagen mRNA, which were also identified in our differential display screening, that show that levels of these transcripts are significantly elevated in the OA-derived samples used in these studies, a finding that is consistent with previous reports of induced collagen synthesis in remodeling OA cartilage (17).
To confirm the differential expression of human HtrA protein  in OA cartilage, high-salt extracts of OA and control cartilage samples were tested by immunoblot analysis using an antiserum prepared against the HtrA domain expressed in E. coli (Fig. 4B). These data reveal a striking difference in the levels of HtrA protein detected in OA versus nonarthritic cartilage extracts, results that are consistent with the analysis of the mRNA levels.
Expression and Proteolytic Activity of ORF480-encoded Protein-A cDNA segment containing the entire coding region of ORF480 was inserted into expression vector pcDNA3 (see "Experimental Procedures" and Fig. 2). This construct directed the expression of the expected size protein (M r ϳ50,000) in an in vitro transcription/translation system (Fig. 5A, lanes 1 and 2). Lower molecular weight products (M r ϳ40 -45,000) were also evident in the gel electrophoresis pattern, a pattern that was remarkably similar to that observed for E. coli HtrA (11,18). Converting the conserved active site serine 328 in ORF480 to alanine by site-directed mutagenesis resulted in the elimination of the lower molecular weight proteins (Fig. 5A, lanes 3  and 4). This result, which was previously shown for E. coli HtrA (18), demonstrates that the translation product of ORF480, the human HtrA protein, is a serine protease with autocatalytic activity.
When ORF480 is expressed in baculovirus-infected Sf9 cells (Fig. 5B), the primary translation product (M r ϳ50,000) is detected by immunoblot analysis using anti-HtrA antiserum in the culture medium by 41 h postinfection. A distinct set of lower molecular weight proteins is evident by immunoblot analysis in samples taken at 65 and 73 h postinfection (Fig. 5B,  top panel), which is consistent with the autocatalytic activity of the ORF480-encoded protein (HtrA) observed in the cell-free expression system described above. As discussed below, a high molecular weight protein is observed by immunoblot analysis in the samples taken at 65 and 73 h postinfection. Incubation of the ORF480-encoded protein (HtrA) with ␤-casein results in the generation of specific proteolytic cleavage products of this substrate (Fig. 5B, bottom panel).
To examine the expression of ORF480 in mammalian cells, cultures of 293 cells were transfected with pcDNA3/ORF480, resulting in the synthesis and secretion of the M r ϳ50,000 HtrA protein as determined by immunoblot analysis (Fig. 5C,  top panel). The media from different isolated clones derived from the transfected culture contain endoprotease activity against ␤-casein (Fig. 5C, bottom panel), resulting in at least four distinct fragments of the substrate, which is similar to the pattern observed in the experiment using baculovirus-expressed HtrA shown in Fig. 5B. The level of proteolytic activity in the samples from each clone (Fig. 5C, bottom panel) correlates with the relative amount of immunoreactive HtrA protein observed in the immunoblot (Fig. 5C, top panel). The S328A FIG. 3. ORF480 contains a Kazaltype inhibitor motif within the mac25 homology domain. A, the amino acid sequence alignment of ORF480 and mac25 is shown; identical (vertical line) and similar (:) residues are indicated. The Kazal-type inhibitor motif is outlined by a rectangle. B, multiple sequence alignment of the conserved Kazal-type inhibitor motif within ORF480 (shaded sequence), human and murine mac25 (9,16), follistatin (31), agrin (32), and several known serine protease inhibitors: elastase inhibitor from Anemonia sulcata (33), rhodiin (34), a representative sequence of the ovomucoid third domain (35), tryptase inhibitor from Hirudo medicinalis (36), protease inhibitor from crayfish (37), and human trypsin inhibitor C (38). A composite image is shown from a representative experiment; PCR products were resolved by polyacrylamide gel electrophoresis followed by staining with SYBR TM -Green (Molecular Probes). Band intensities were quantified using a PhosphorImager TM and ImageQuant software (Molecular Dynamics). *, the mean ratio of expression (normalized to actin) in OA cartilage to that in nonarthritic cartilage from multiple (n) experiments using at least three samples from each is indicated. COL3A and COL2A are abbreviations for pro-␣1 (III) and pro-␣1 (II) collagens, respectively. B, detection of ORF480-encoded HtrA protein in high-salt extracts of human OA and nonarthritic cartilage by immunoblot analysis (see "Experimental Procedures"). The anti-HtrA antiserum exhibits low cross-reactivity with serum albumin. A supernatant sample from cultured 293 cells transfected with pcDNA3-ORF480 was included as a positive control (ϩ) in the immunoblot analysis. mutant of ORF480 (HtrA) expressed in transfected 293 cells lacks proteolytic activity against ␤-casein (Fig. 5D) and does not exhibit autoproteolysis (Fig. 6).
Stable Complex of HtrA with ␣ 1 -Antitrypsin-In immunoblots showing ORF480 protein (HtrA) expressed in both 293 cells and in baculovirus-infected Sf9 cells, high molecular weight bands (M r Ͼ 120,000) were evident (indicated with asterisks in Fig. 5, B and C). Amino acid sequence analysis (Edman degradation) of the protein isolated from this fraction indicated a 1:1 ratio of HtrA sequence (XAPLAAGXPDRXEPA, residues 30 -44) and the amino-terminal sequence of the ma-ture form of ␣ 1 -antitrypsin (XVLQGHAVXE). Results from additional expression experiments demonstrated that the stable complex observed in cultures incubated in the presence of serum was not detected in serum-free conditions (Fig. 6). In addition to the indicated protein band (*) migrating above the 121-kDa standard, an immunoreactive band around 78 kDa appears after 48 -72 h of culture incubation, which is probably a degradation product of the complex. The addition of purified ␣ 1 -antitrypsin to cultures incubated in serum-free medium resulted in the formation of the high molecular weight proteins with the same electrophoretic mobility as those observed in cultures with serum. The formation of the stable HtrA/␣ 1antitrypsin complex does not occur when the active site serine 328 in HtrA is mutated to alanine (Fig. 6).
The Mammalian HtrA Gene Is Evolutionarily Conserved-In anticipation of experiments to study the regulation of ORF480 (HtrA) in animal models of OA, we sought to determine whether the human cDNA could be used as a probe to screen for homologous cDNA clones from other mammalian species. Fig.  7A shows a genomic ZOO-BLOT analysis in which total EcoRIdigested genomic DNA isolated from various mammalian and one avian species was hybridized with 32 P-labeled human cDNA under relatively high stringency (see "Experimental Procedures"). The results, which show specific hybridization to a limited number of genomic DNA fragments, suggest that the gene sequence coding for the HtrA-related domain of ORF480 is conserved among vertebrate species. This interpretation was confirmed by analysis of the nucleotide sequences of partial ORF480 cDNA clones isolated from cow, guinea pig, and rabbit, which are 91, 89, and 88% identical to the human sequence, respectively (GenBank accession numbers AF097707, AF097706, AF097708, and AF097709). Moreover, the amino acid sequences derived from the respective cDNA sequences of the three mammalian species are 98% identical to the human sequence (Fig. 7B).

DISCUSSION
As part of an ongoing effort to profile alterations in gene expression in osteoarthritic cartilage, we identified and characterized a cDNA, referred to in this report as ORF480, and its translation product. The data reported here indicate that the levels of ORF480 mRNA and protein are significantly elevated in osteoarthritic cartilage. ORF480 encodes a protein with two distinct domains of homology. The amino-terminal domain of ORF480 is homologous to mac25, a recently characterized gene product related to insulin-related growth factor-binding protein (9) and follistatin (16). The second domain, comprising the majority of the ORF480 amino acid sequence, is greater than 40% identical to bacterial HtrA serine protease. The evidence presented here demonstrates the in vitro proteolytic activity of the ORF480-encoded protein, which we conclude is the human homologue of bacterial HtrA.
In bacteria, HtrA is a critical component of the universal cellular response to stress, which is characterized by the induc-tion of a set of so-called heat shock proteins (19). In addition to temperature elevation, heat shock proteins are induced by oxidative stress (20), viral (phage) infection (21), and intracellular expression of aberrant proteins (22). A functional htrA (high temperature requirement) gene is indispensable for the bacterial cell to survive heat shock (11). HtrA is identical to DegP FIG. 7. Mammalian HtrA is highly conserved. A, human ORF480 cDNA hybridizes to genomic DNA of several species. A ZOO-BLOT membrane filter (CLONTECH) containing EcoRI-digested genomic (4 g DNA/lane) from different vertebrate species (indicated above each lane) was hybridized with the HtrA-related segment of ORF480 as described under "Experimental Procedures." B, amino acid sequence alignment of human HtrA with other mammalian homologues. The translated amino acid sequence of ORF480 (single-letter code) is shown with residue numbers indicated to the right. An alignment with E. coli HtrA is shown above the human sequence with the residue numbers indicated at the ends of each homology segment. Identical amino acid sequences between ORF480-encoded protein and human mac25 (see Fig. 3) are indicated (*) above the ORF480 residues. The 16 cysteine residues in the amino-terminal domain of ORF480 are underlined. The conserved serine protease active site sequence (39) is outlined by a rectangle, and the active site serine residue is shaded. The putative active site histidine (His-220) and aspartate (Asp-250) residues that comprise the remainder of the catalytic triad are also indicated. Identical residues between human, cow, rabbit, and guinea pig HtrA homologues are denoted with a hyphen, and the actual amino acid substitutions in each species are shown. (23), a serine endoprotease originally named "Do" as one of several proteolytic activities purified from E. coli (24). Mutation of the active site serine 236 to alanine in HtrA results in a loss of protease activity in vitro and a loss of function, as determined by the inability to suppress the thermosensitivity of htrA-null mutants (11). Lipinska et al. (11) suggested that the role of HtrA is proteolytic cleavage of toxic denatured proteins in the periplasmic space. Additional experimental evidence supporting this hypothesis has been recently reported by other investigators (25,26).
Based on the conservation of the HtrA (ORF480) sequence as shown in this report (at the DNA level in mammals and at the amino acid level in bacteria), HtrA function may also be conserved; however, any definitive description of its biological role will require further investigation. Identification of physiologically relevant substrates for the human serine protease encoded by ORF480 will be an important aspect of those studies. Possible substrates of interest include other proteases, extracellular matrix proteins, growth factors, and proteins that modulate growth factors. The autocatalytic cleavage of human HtrA protein that we observed in vitro may reflect a biologically significant mode of regulation in vivo, because at least one processed form of HtrA is detected by immunoblot analysis in OA cartilage. The finding that ␣ 1 -antitrypsin binds to and forms a stable complex with HtrA suggests that this serine protease inhibitor may play a role in its regulation in vivo. As one would anticipate, the proteolytic activity of human HtrA is inhibited by 1-2 M ␣ 1 -antitrypsin. 6 Levels of various protease inhibitors, including ␣ 1 -antitrypsin, are known to be altered in osteoarthritic cartilage (27). Additional experimental evidence will be required to address the intriguing aspects of how human HtrA expression and activation are regulated.
The high degree of conservation of mammalian HtrA and the addition of a new functional domain during evolution, namely mac25, suggest a biological role for the ORF480-encoded protein (HtrA) beyond that of processing denatured proteins. Although mac25 was initially described as a member of the IGFbinding protein family (9), Kato et al. (16) noted that mac25 is more closely related to follistatin, an activin-binding protein.
They demonstrated that its expression in transfected osteosarcoma cells results in clonal growth inhibition. A similar phenomenon was consistently observed during the process of isolating clones of human 293 cells transfected with ORF480 cDNA. 7 It is possible that human HtrA is involved in cell growth regulation, perhaps via a modulation of growth factor systems other than IGF, e.g. the activin/inhibin system (28).
The Kazal-type inhibitor motif in ORF480-encoded protein, which is conserved in a diverse group of serine protease inhibitors, also occurs within mac25, follistatin, and agrin. Agrin and agrin-related proteins appear to function as extracellular components that bind to and regulate the activity of growth factors (29). Recombinant agrin has been shown to inhibit serine proteases of the trypsin class but not the thrombin class (30). The presence of the protease inhibitor motif in ORF480 suggests that the human HtrA serine protease may be a selfregulating enzyme; however, the alternative explanation -that it may regulate other serine proteases -should not be excluded.
The experimental results reported here demonstrating the in vitro activities of human HtrA, together with clues derived from the nature of the various proteins to which it is related, will guide future experimental approaches to reveal the (patho)physiological role of mammalian HtrA.