Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase.

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.

A human-specific polymorphism in the coding region of the aggrecan gene. Variable number of tandem repeats produce a range of core protein sizes in the general population.

Aggrecan, one of the major structural genes of cartilage, encodes a proteoglycan core protein composed of an extended central glycosaminoglycan-bearing domain, flanked by globular domains at each end. The central region consists of long stretches of repeating amino acids that serve as attachment sites for glycosaminoglycans such as chondroitin and keratan sulfate; the terminal globular domains interact with other cartilage components. The glycosaminoglycan attachment region is encoded in several species by a single large exon, within which are several different types of repeating sequences. Several species show within this exon a similar block of conserved repeats for attachment of chondroitin sulfate, but in humans this group of repeats is particularly well conserved. Examination of genomic DNA from a population of unrelated individuals by polymerase chain reaction or Southern blot assays shows this block of repeat sequences exists in multiple allelic forms, which differ by the number of repeats at this site in each allele. Thirteen different alleles have been identified, with repeat numbers ranging from 13 to 33. This is an unusual example of an expressed variable number of tandem repeat polymorphism. This polymorphism is apparently restricted to humans, of several species examined. This polymorphism results in individuals with differing length aggrecan core proteins, bearing different numbers of potential attachment sites for chondroitin sulfate. The possibility exists for a molecular understanding of biological variation in cartilage functional properties.

Alternative exon splicing within the amino-terminal nontriple-helical domain of the rat pro-alpha 1(XI) collagen chain generates multiple forms of the mRNA transcript which exhibit tissue-dependent variation.

Type XI collagen is an integral, although minor component of cartilage collagen fibrils. We have established that alternative exon usage is a mechanism for increasing structural diversity within the amino-terminal nontriple helical domain of the pro-alpha 1(XI) collagen gene. cDNA clones spanning the amino-terminal domain were selected from a rat chondrosarcoma library, and were shown to contain two major sequence differences from the previously reported human sequence. The first difference was the replacement of sequence encoding an acidic domain of 39 amino acids in length by a sequence encoding a 51-amino acid basic domain with a predicted pI of 11.9. The second difference was the absence of a sequence that would translate into a highly acidic 85-amino acid sequence downstream from the first variation. These two changes, expressed together, result in the replacement of most of the acidic domain with one that is smaller and basic. These two sequence differences serve to identify subdomains of a variable region, designated V1 and V2, respectively. V1a is defined as the acidic 39-amino acid sequence element and V1b is defined as the 51-amino acid basic sequence. Analysis of genomic DNA revealed that both V1a and V1b are encoded by separate adjacent exons in the rat genome and V2 is also encoded in a single exon downstream. Analysis of mRNA from cartilage-derived sources revealed a complex pattern of alpha 1(XI) transcript expression due to differential exon usage. In non-cartilage sources, the pattern is less complex; the most prevalent form is the one containing the two acidic sequences, V1a and V2.

The structure of the rat aggrecan gene and preliminary characterization of its promoter.

Aggrecan is a major structural component of cartilage extracellular matrix and a specific gene product of differentiated chondrocytes. cDNA clones have been used to isolate rat aggrecan genomic clones from phage and cosmid libraries, producing over 80 kilobases (kb) of overlapping DNA containing the complete rat aggrecan gene, including 12 kb of 5'- and 8 kb of 3'-flanking DNA. DNA sequencing shows 18 exons, most of which encode structural or functional modules; exceptions are domains G1-B and G2-B, which are split into two exons and the G3 lectin domain, which is encoded by three exons. There is one expressed epidermal growth factor-like exon and in addition a non-expressed "pseudo-exon" encoding a heavily mutated epidermal growth factor-like domain. Intron sizes have been determined by restriction mapping and inter-exon polymerase chain reaction; a 30-kb intron separates exons 1 and 2. Exon 1 has been mapped by primer extension and S1 nuclease protection; it encodes 381 base pairs (bp) of 5'-untranslated sequence. There is a minor promoter which initiates transcription an additional 68 bp 5' of the major promoter start site. DNA sequence is reported for a 529-bp fragment encompassing exon 1, including 120 bp of 5'-flanking DNA comprising the promoter. This promoter is lacking the TATAA or CCAAT elements but has several putative binding sites for transcription factors. A 922-bp DNA fragment with 640-bp 5'-flanking DNA and 282-bp exon 1 sequence showed higher promoter activity in transfected chondrocytes than in fibroblasts, is completely inactive in the reverse orientation, and is strongly enhanceable in the forward direction by the SV40 enhancer.

Complete coding sequence, deduced primary structure, chromosomal localization, and structural analysis of murine aggrecan.

We have isolated and sequenced overlapping cDNA clones encoding the entire core protein of aggrecan (the large aggregating chondroitin sulfate/keratan sulfate proteoglycan of cartilage) from three chondrocyte cDNA libraries of BALB/c mice and localized the aggrecan gene in mouse chromosome 7. We determined 7386 bp of the cDNA sequence, including 132 and 854 nucleotides of 5' and 3' untranslated regions, respectively. The core protein precursor is 2132 amino acids long (M(r) 222,008), including a 19-residue secretory signal peptide. The overall amino acid sequence of the mouse aggrecan shows 91.6% identity to rat and 72.5% to human aggrecan. Comparison of the amino acid sequences of various domains and subdomain structures of mouse aggrecan to known sequences of other species and related proteins (versican, neurocan, link protein, and lymphocyte homing receptor CD44) revealed high levels of identity of the G1, G2, and G3 globular domains and relatively less conserved structures in the interglobular and glycosaminoglycan-attachment regions. Epidermal growth factor (EGF)-like module was detected in only a minor fraction of aggrecan clones, while the complement regulatory protein (CRP)-like domain was regularly expressed in all samples.

Characterization of type II and type XI collagen synthesis by an immortalized rat chondrocyte cell line (IRC) having a low level of type II collagen mRNA expression.

The biosynthesis of type XI and type II collagens was examined using a stable rat chondrocyte cell line established by W. E. Horton et al. (1988, Exp. Cell Res. 178, 457-468.). These cells (IRC; immortalized rat chondrocytes) were created by transformation with a murine retrovirus carrying the v-myc and v-raf oncogenes. They grow in suspension culture as multicellular aggregates and synthesize typical cartilage proteins, aggrecan and link protein. Type II collagen is absent or synthesized at severely reduced levels, as shown by Northern analysis of mRNA. Thus, this cell type represents a unique model in which to study cartilage matrix protein interactions in the absence of type II collagen. A more detailed look at the proteins secreted into the medium by metabolically labeled IRC cells revealed the presence of collagenase-sensitive bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were identified as the alpha 1, alpha 2, and alpha 3 chains of heterotrimeric type XI collagen by electrophoretic migration after pepsin digestion, by CNBr peptide mapping, and by immunoprecipitation with antibodies to rat alpha 1(XI). mRNA for all three chains was detected by Northern blot analysis. The data indicate that the low level of alpha 1(II) mRNA previously detected in these cells is translated into pro alpha 3(XI) polypeptide chains which are incorporated into molecules of type XI. Under normal culture conditions, homotrimers of type II collagen were not detected. The carboxyl propeptide domain of the fibrillar collagens directs chain selection and molecular assembly of the trimeric molecules. The sequence of the carboxyl propeptide domain from pro alpha 3(XI) of IRC cells was found to be identical to this domain from pro alpha 1(II) of swarm rat chondrosarcoma, supporting previous evidence that pro alpha 3(XI) and pro alpha 1(II) have the same primary structure. When cultured in the presence of 50 mM arginine, IRC cells could be induced to synthesize pro alpha 1(II) chains in excess over pro alpha 1(XI) and pro alpha 2(XI). Only under these conditions were type II collagen molecules detected, suggesting a preferential association of pro alpha 1(II) with the pro alpha 1 and/or pro alpha 2 chains of type XI collagen.

Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms.

We have obtained the complete coding sequence of the large aggregating chondroitin sulfate proteoglycan of human cartilage (aggrecan) from a combination of cDNA and genomic exon sequencing. We screened a human costal chondrocyte cDNA library, using rat aggrecan cDNA probes, and obtained three nonoverlapping clones totaling 6.2 kilobases in length. These clones were sequenced, and the sequence of the gaps between clones was obtained from genomic exon fragments and polymerase chain reaction-amplified cDNA. The composite sequence is 7137 nucleotides long, encoding 2316 amino acids. The human and rat aggrecan amino acid sequences are about 75% identical, with domains ranging from 100% to about 60% of conserved amino acids. The human sequence contains two regions of highly conserved repeats not found in rat aggrecan: 11 repeats of a hexameric sequence in the keratan sulfate attachment domain, E-E-P-(S,F)-P-S; and a 19-amino acid sequence reiterated 19 times, in the CS-1 portion of the serine-glycine-containing region. There are at least three forms of aggrecan transcripts, generated by alternative exon usage, and the form reported here is the shortest and also the most prevalent, lacking both the epidermal growth factor-like domain, and the complement regulatory protein-like sequence.

Folding of carboxyl domain and assembly of procollagen I.

An early form of procollagen I was found in acetic acid extracts of radioactively labeled chick embryo skull bones. It resembled native procollagen I, but sedimented slightly faster, and its component chains were slightly underhydroxylated and were not disulfide-linked to each other, although its propeptides were internally disulfide-bonded. Pulse-chase experiments showed its conversion to disulfide-linked procollagen. As the same conversion occurred when proline hydroxylation was blocked by 2,2'-dipyridyl, we infer that the formation of this precursor from its component chains does not require collagen triple helix formation. We suggest that interaction between the folded carboxyl propeptides of individual pro-alpha (I) chains is an important step in the formation of this precursor and of procollagen I. Studies of the refolding and association of fully reduced and denatured carboxyl propeptides supported this concept. In the presence of glutathione the correct disulfide bonds could be reestablished, as judged by a mapping of some tryptic peptides. Individual carboxyl propeptides refolded first, and this occurred even in 2 M urea. Recognition between folded carboxyl propeptides occurred only when less than 0.5 M urea was present. The presence of the carboxyl telopeptides was important for trimeric reassembly. Individual propeptides also folded spontaneously during cell-free translation of pro-alpha (I) chains and were recognized by specific antibodies. We consider the role of carboxyl propeptides in the formation of procollagen I molecules and suggest a model of self-assembly, possibly facilitated by interactions with the luminal surface of the rough endoplasmic reticulum.

Biosynthesis of collagen.

During the biosynthesis and assembly of collagen structures, disulfide links can serve several functions. During biosynthesis they successively stabilize intrapeptide folding and associations of three chains into one molecule. Studies on the refolding and reassociation of reduced and denatured carboxyl propeptides of procollagen I showed that successive interactions of folding and assembly are successively weaker. Disulfide bridges were reestablished within correctly refolded carboxyl propeptides. Rearrangements of disulfide bridges may occur during the processing of type V procollagen molecules as these collagens become incorporated into extracellular matrix. The basement membrane procollagen IV molecules become disulfide linked at each end into networks, and there are indications that further rearrangements of disulfide links may allow additional modulation.

Structural implications from an electronmicroscopic comparison of procollagen V with procollagen I, pC-collagen I, procollagen IV, and a Drosophila procollagen.

Chick embryo procollagen V, procollagen I, and pC-collagen I were sprayed, rotary shadowed, and compared electronmicroscopically with mouse procollagen IV and Drosophila procollagen produced by cell cultures and prepared in the same way. All the molecules appeared as threads and had a prominent knob protrusion at one end. For procollagen I and pC-collagen I this must correspond to the carboxyl propeptides, and most likely corresponds to the similarly sized carboxyl propeptides of procollagen IV. Procollagen V appeared as a thread of nearly the same length as procollagen I but with substantial knobs at both ends, corresponding to its known propeptides. The length of procollagen V and the known, successive processing of its propeptides make procollagen V more similar to the interstitial collagens than to the basement membrane procollagen IV, which is 1.4 times as long and has so far been found not to be processed. Drosophila procollagen resembles procollagen IV in length and appearance.

Qualitative evaluation of macroscopic flocculation tests for syphilis.

Five thousand two hundred and two sera were tested in parallel using the Rapid Plasma Reagin (RPR) Card Test, the Reagin Screen Test (RST), and the Venereal Disease Research Laboratory (VDRL) Slide Test. The fluorescent treponemal antibody absorption (FTA-ABS) test was then used to confirm the reactive specimens. This comparison study was to evaluate the RPR and the RST when used in a screening program. Of the total 5,202 samples, 199 (3.8%) were VDRL reactive, 187 (3.6%) were RPR reactive, and 183 (3.5%) were RST reactive. Of the 199 VDRL reactive, 116 (58.2%) were confirmed by the FTA-ABS, 137 (73.3%) of the 187 RPR reactives were confirmed by the FTA-ABS and of the 183 RST reactives, 115 (62.8%) were confirmed by the FTA-ABS. Included in this discussion are some testing and procedural problems encountered during the study.