Abstract
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.
Highlights
Sequence shows that the 104-amino acid mature core The 104-amino acid core protein contains a 48-amino acid protein is processed from a 179-aminoacid prepro core segment that consists entirely of the dipeptide serine-glycine protein precursorwhich, in addition to the mature correepeated 24 times
Further analysis of the PG19 mRNA by RNA internal signal sequence or that thetranslation initiation site 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
We report here the identification and sequencingof a cDNA clone that reveals the existence of a PG19 prepro core protein containinga signal sequence as well as a propeptide
Summary
Tein sequence of P G 1 9 and provide evidence of polypeptide processing during thebiosynthesis of the proteoglycan core protein. CDNA Clones”PG19 cDNA clones pPGl and pPG6 were isolated from an L2 cell cDNA library using mixed oligonucleotides derived from the NHz-terminal aminoacid sequence of the mature proteogly-. Proteoglycans are protein glycoconjugates composed of a core protein with glycosaminoglycan covalently attached to it. They are abundant constituentsof extracellular matrices, can (2). The Maxam-Gilbert functions of proteoglycans are poorly understood, and there procedure (8) was used to sequence part of the cDNA. This article must be hereby hybrid-selectedusing cloned PG19 cDNA (9). Hybrid-selected mRNA was in vitro translated using a rabbit reticulocyte lysate (New England Nuclear), and translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE’) using 15%gels with a 4% stacking gel.
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