Occurrence of PG-Lb, a leucine-rich small chondroitin/dermatan sulphate proteoglycan in mammalian epiphyseal cartilage: molecular cloning and sequence analysis of the mouse cDNA.

PG-Lb is a chondroitin/dermatan sulphate proteoglycan first isolated from chick embryo limb cartilage. It had been assumed that osteoglycin represents its mammalian homologue. However, partial amino acid sequences of a novel proteoglycan from bovine epiphyseal cartilage showed high identity with those of chick PG-Lb (P. Neame, L. Rosenberg and M. Höök, personal communication). Reverse transcriptase PCR using degenerate oligonucleotide primers gave a cDNA fragment that might correspond to mouse PG-Lb. We isolated a clone from a cDNA library of newborn mouse epiphyseal cartilage using the cDNA fragment as a probe. The cloned cDNA was 1430 bp long and contained a 966 bp open reading frame which encoded the core protein consisting of 322 amino acid residues. The deduced amino acid sequence showed a high overall identity with chick PG-Lb (about 62%, reaching about 80% over the carboxyl two-thirds). In addition, the amino acid sequence contained a signal peptide, six cysteine residues at the invariant relative position to chick PG-Lb, six leucine-rich repeats at the carboxyl two-thirds, three possible glycosaminoglycan-attachment sites (two sites at the N-terminal side and one site at the C-terminus) and two possible Asn-glycosylation sites near the C-terminus. Northernblot analysis demonstrated the specific expression of a 1.5 kb message in cartilage and testis. These structural features and the characteristic expression suggest that the cloned molecule is mouse PG-Lb.

2B1 antigen characteristically expressed on extracellular matrices of human malignant tumors is a large chondroitin sulfate proteoglycan, PG-M/versican.

2B1 is a monoclonal antibody against a large proteoglycan isolated from human yolk sac tumor (M. Sobue et al., Histochem. J., 21: 455-460, 1989). The antigen is expressed in a variety of embryonal tissues as well as most if not all malignant tumor tissues. However, the expression in normal adult tissues is limited to some tissues, such as the smooth muscle layers of the aorta. We characterized the 2B1 antigen isolated from the conditioned medium of human malignant fibrous histiocytoma and found that immunological and biochemical properties are identical to those of a large chondroitin sulfate proteoglycan, PG-M/versican. Partial amino acid sequences of peptides obtained from the core protein by V8 protease digestion and subsequent SDS-PAGE were detected in the reported amino acid sequence of human PG-M/versican with a complete identity. Furthermore, 2B1 was distinctly reactive to the expressed protein by transfection of the cDNA for the shortest form into mouse cells. The results indicate that the antigen is the PG-M core protein, and the epitope may be in one of the globular domains. It is thus likely that PG-M/versican is one of the extracellular matrix components characteristic of human malignant tumors.

Molecular cloning and expression of chick chondrocyte chondroitin 6-sulfotransferase.

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. The enzyme has been purified previously to apparent homogeneity from the serum-free culture medium of chick chondrocytes. The purified enzyme also catalyzed the sulfation of keratan sulfate. We have now cloned the cDNA of the enzyme. This cDNA contains a single open reading frame that predicts a protein composed of 458 amino acid residues. The protein predicts a Type II transmembrane topology similar to other glycosyltransferases and heparin/heparan sulfate N-sulfotransferase/N-deacetylases. Evidence that the predicted protein corresponds to the previously purified C6ST was the following: (a) the predicted sequence of the protein contains all of the known amino acid sequence, (b) when the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, both the C6ST activity and the keratan sulfate sulfotransferase activity were overexpressed, (c) a polyclonal antibody raised against a fusion peptide, which was expressed from a cDNA containing the sequence coding for 150 amino acid residues of the predicted protein, cross-reacted to the purified C6ST, and (d) the predicted protein contained six potential sites for N-glycosylation, which corresponds to the observation that the purified C6ST is an N-linked glycoprotein. The amino-terminal amino acid sequence of the purified protein was found in the transmembrane domain, suggesting that the purified protein might be released from the chondrocytes after proteolytic cleavage in the transmembrane domain.

Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing.

We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K. (1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.

Molecular cloning of rat RAC protein kinase alpha and beta and their association with protein kinase C zeta.

cDNAs of RAC protein kinases (RAC-PK) alpha and beta were cloned from a rat testis cDNA library. The predicted open reading frames encode 480 and 481 amino acids of RAC-PK alpha and beta, respectively, and the rat RAC-PK alpha and beta have sequences conserved among different mammalian species such as the pleckstrin homology domain at their amino-terminal region and the protein-serine/threonine kinase catalytic domain at their carboxyl-terminal region. RNA blot analysis showed wide distribution of two RAC-PK in rat tissues. Immunoprecipitation analysis revealed that RAC-PK alpha and beta associate with protein kinase C zeta through the pleckstrin homology domain in vitro, suggesting the interaction between RAC-PK and protein kinase C.