Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment

Peptides were derived from the large chondroitin sulfate proteoglycan from chick cartilage by clostripain digestion. Using differential chondroitinase ABC and keratanase treatment and direct carbohydrate analysis, three major peptides of 86, 75, and 27 kDa were shown to bear only chondroitin sulfate chains. Another major peptide of 65 kDa was shown to contain both chondroitin sulfate and keratan sulfate chains, allowing it to be separated from the peptides derived from the chondroitin sulfate domain by DEAE-cellulose chromatography. An additional new peptide (100 kDa) containing keratan sulfate chains was found only in clostripain digests of proteoglycan-hyaluronate-link protein aggregates. Unlike any of the other peptides derived from clostripain digestion of proteoglycan monomer or aggregate, this peptide had the properties of a functional hyaluronate binding region. All of these peptides were purified to apparent homogeneity by preparative electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and deglycosylated with anhydrous hydrogen fluoride. Automated Edman degradation of the two largest chondroitin sulfate peptides revealed that they had unique N termini and several unrecognized residues, which were all subsequently revealed to be modified serine residues following deglycosylation. The keratan sulfate-bearing peptide also had a unique N terminus, which contained a single unrecognized residue, even after HF deglycosylation. Finally, the N terminus of the hyaluronate binding region was blocked. These studies allow estimates of core peptide masses in the absence of carbohydrate as well as provide primary amino acid sequence for O-xylosylated serine residues in the multiply substituted proteoglycans.     

Gene structure of chick cartilage chondroitin sulfate proteoglycan (aggrecan) core protein

Large aggregating chondroitin sulfate proteoglycan (CSPG/aggrecan) is one of the major extracellular matrix components in cartilage. The core protein is also large, over 200 kDa, and modular with a distinct correspondence between protein structural domains and the encoding exons. Here we report the isolation, using chick CSPG cDNA probes and the ensuing sequencing, of genomic clones containing exons encoding the chick CSPG core protein. The 5 two globular domains, G1 and G2, are encoded by four and three exons, respectively, and the interglobular domain is encoded by a single exon. The chondroitin sulfate attachment domain is encoded by the largest exon, 3,216 bp, which is approximately 50% of the total coding sequence. Combined with the previous report (Tanaka, T., Har-el, R. Tanzer, M.L. 1988 J. Biol. Chem. 263, 15831–15835), these data reveal that the chick CSPG gene contains at least 18 exons spanning a genome which is greater than 30 kb. No evidence was obtained for multiple genes for aggrecan in the chick genome. Elucidation of the chick genomic structure allows comparison of the avian and mammalian link protein genes to the homologous portions of avian and mammalian core protein genes (hyaluronate binding domain) with respect to their origins and paths of duplication and divergence. Correspondence to: N.B. Schwartz     

Identification from cDNA of the precursor form of a chondroitin sulfate proteoglycan core protein

The yolk sac carcinoma cell line L2 secretes a chondroitin/dermatan sulfate proteoglycan that has an Mr 10,000 core protein and carries an average of 14 glycosaminoglycan chains. The amino acid sequence of the mature core protein has been determined from cloned cDNA (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1321-1325). From additional cDNA sequences described in this report we have identified the prepro core protein precursor of the yolk sac carcinoma chondroitin/dermatan sulfate proteoglycan. From the amino acid sequence of the core protein precursor can be deduced the protein processing events in the biosynthesis of the proteoglycan. The amino acid sequence shows that the 104-amino acid mature core protein is processed from a 179-amino acid prepro core protein precursor which, in addition to the mature core protein, contains a 26-amino acid signal peptide as well as a 49-amino acid propeptide. The molecular weight of the prepro core protein predicted from the cDNA sequence (Mr = 18,600) was in good agreement with the molecular weight of the in vitro translation product (Mr = 19,000) of hybrid-selected mRNA. Accordingly, we have designated the proteoglycan core protein PG19. Further analysis of the PG19 mRNA by RNA sequencing confirmed the identification of the core protein translation initiation codon by revealing stop codons in all three reading frames of the upstream mRNA sequence. Primer extension analyses demonstrated that the 5' untranslated sequence of the proteoglycan mRNA is approximately 220 nucleotides in length, which, combined with the length of cDNA clones, accounts for the entire length of the coding sequence of PG19 mRNA from L2 cells. The cDNA sequences presented here establish the complete protein sequence of PG19 and provide evidence of polypeptide processing during the biosynthesis of the proteoglycan core protein.     

cDNA clone to chick corneal chondroitin/dermatan sulfate proteoglycan reveals identity to decorin.

A 1.6-kb cDNA clone was isolated by screening a library prepared from chick corneal mRNA with a cDNA clone to bovine decorin. The cDNA contained an open reading frame coding for a M(r) 39,683 protein. A 19-amino-acid match with sequence from the N-terminus of core protein from the corneal chondroitin/dermatan sulfate proteoglycan confirmed the clone as a corneal proteoglycan and the homology with human and bovine decorin confirmed its identity as decorin. Structural features of the deduced sequence include a 16-amino-acid signal peptide, a 14-amino-acid propeptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 63% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I. Chick decorin contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The presence of beta bend amino acids flanking the units may serve to delineate the units as structural elements of the leucine-rich region. Sequence homology within the repeats and the spacing of the repeats suggest that this region arose by duplication. Chick decorin primarily differs from mammalian decorins in the 19-amino-acid sequence that starts the N-terminus of the core protein. Within this region, the serine that serves as a potential acceptor for the chondroitin/dermatan sulfate side chain is preceded by a glycine instead of being followed by a glycine as it is in the mammalian decorins and all other mammalian proteoglycans.     

Regulation of chondroitin sulfate synthesis. Effect of beta-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains, and core protein

Monolayer cultures of embryonic chick chondrocytes were incubated with 35SO42- in the presence and absence of 1.0 mM p-nitrophenyl-beta-d-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of beta-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both beta-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible for the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, beta-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the beta-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.     

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.     

Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia.

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.     

A fibroblast chondroitin sulfate proteoglycan core protein contains lectin-like and growth factor-like sequences

We have isolated cDNA clones that code for a proteoglycan-related polypeptide with unique properties. A lambda gt11 expression library made from human fibroblast mRNA was screened with an antiserum made against a proteoglycan fraction from human fetal membranes. One group of positive clones revealed an open reading frame coding for 685 amino acids from the COOH terminus of a polypeptide. This amino acid sequence contains a domain that is strongly homologous with the COOH-terminal core protein domain of the large aggregating cartilage proteoglycan. This domain also contains sequences that are homologous with vertebrate lectins that bind terminal galactosyl, N-acetyl-glucosaminyl or mannosyl residues. On the NH2-terminal side of the lectin-like domain the cDNA-derived amino acid sequence contains two epidermal growth factor-related segments. The cDNA clones were shown to belong to a chondroitin sulfate proteoglycan by using antisera made against two peptides predicted from the cDNA sequence. These antisera were reactive with a proteoglycan fraction from fibroblasts after chondroitinase treatment of the fraction but not after treatment with heparinase or no treatment. Among the several polypeptides reactive with the anti-peptide antibodies the largest one, corresponding to a molecular weight of about 400,000, is likely to be the intact core protein, whereas the smaller polypeptides may be processing products or products of artifactual proteolysis. These results show that the amino acid sequence belongs to a proteoglycan core protein, and the sequence, therefore, provides a molecular definition to this proteoglycan. The lectin-related and growth factor-like sequences in the core protein of this proteoglycan suggest that it may play a role in intercellular signaling.     

Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage.

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.     

Kinetics of intracellular processing of chondroitin sulfate proteoglycan core protein and other matrix components

Pulse-chase labeling techniques are used in conjunction with subcellular fractionation and quantitative immunoprecipitation to define the kinetics of intracellular translocation and secretion of proteoglycan core protein, along with link protein and type II collagen. In embryonic chick chondrocytes the core protein is processed very rapidly, exhibiting a t 1/2 in both the rough endoplasmic reticulum and golgi region of less than 10 min. Link protein appears to be processed as rapidly as the core protein, but the kinetics of type II collagen secretion is 3-4 times slower. These results are consistent with possible segregation and coordinate intracellular processing of link protein and core protein, macromolecules which are known to associate extracellularly. In contrast, rat chondrosarcoma chondrocytes translocated and secreted the core protein much more slowly (t 1/2 = 40 min) than the chick cells, perhaps due to the significantly reduced levels of galactosyltransferase I observed in the transformed chondrocytes.     

Analysis of the chondroitin sulfate proteoglycan core protein (CSPGCP) gene in achondroplasia and pseudoachondroplasia.

Achondroplasia and pseudoachondroplasia are autosomal dominant skeletal dysplasias resulting in short-limbed dwarfism. Histologic and ultrastructural studies of the cartilage in pseudoachondroplasia and in homozygous achondroplasia have suggested a structural abnormality in chondroitin sulfate proteoglycan (CSPG), a major structural protein in the extra-cellular matrix. The gene encoding CSPG core protein (CSPGCP) is thus a logical "candidate gene" for analysis in these conditions. cDNA probes encoding CSPGCP were used to identify restriction fragment length polymorphisms (RFLPs) in DNA from a panel of control individuals. No gross alterations at the CSPGCP locus were noted in DNA from 37 individuals with achondroplasia and 5 individuals with pseudoachondroplasia. In addition, allelic frequencies of the RFLPs were not significantly different among controls and patients with either condition. In one three-generation family with achondroplasia, close linkage of the CSPGCP locus and the skeletal dysplasia was excluded using a Bgl II polymorphism. Similarly, in a three-generation family with pseudoachondroplasia, the CSPGCP gene was not tightly linked to the disease phenotype. These results indicate that mutations at the chondroitin sulfate proteoglycan core protein locus do not cause achondroplasia or pseudoachondroplasia in these families.     

Nucleotide sequence of a cDNA clone encoding mouse transition protein 1

We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455–464; Hecht et al. (1986) Exp. Cell Res. 164, 183–190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.