Molecular characterization of a novel basement membrane-associated proteoglycan, leprecan.
A monoclonal antibody was used in early studies to identify a novel chondroitin sulfate proteoglycan, secreted by L-2 cells, the core protein of which was approximately 100 kDa. To characterize this proteoglycan core protein at the molecular level, an L-2 cell cDNA library was probed by expression screening and solution hybridization. Northern blot analysis assigned transcript size to approximately 3.1 kilobases and, after contig assembly, the coding region of the mRNA corresponded to 2.18 kilobases. Immunoassays were performed to confirm the identity of this sequence, using a polyclonal antibody raised against an expressed fusion protein encoded by sequence representing the carboxyl half of the molecule. The antibody recognized the core protein in Western blots after prior digestion of the intact proteoglycan with chondroitinase ABC. Immunostaining tissue sections with the same antibody localized the proteoglycan to basement membranes, and expression of the entire sequence in Chinese hamster ovary K-1 cells showed that the protein encoded by the sequence secreted as a chondroitin sulfate proteoglycan. The core protein not only has motifs permitting glycosylation as a proteoglycan, but also possesses the endoplasmic reticulum retrieval signal, KDEL, which suggests that, in addition to its role as a basement membrane component, it may also participate in the secretory pathway of cells.
Wassenhove-McCarthy
D J
DJ
Department of Pathology, School of Medicine, Louisiana State University Medical Center, Shreveport, Louisiana 71130, USA.
McCarthy
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001485:Basement Membrane / Q000737:chemistry*; D016466:CHO Cells; D019765:Chondroitin ABC Lyase / Q000378:metabolism; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D003871:Dermatan Sulfate / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D007678:Kidney Glomerulus / Q000166:cytology; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D064828:Prolyl Hydroxylases; D017433:Protein Structure, Secondary; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics; D012313:RNA / Q000032:analysis; D051381:Rats; D016415:Sequence Alignment; D014162:Transfection; D014407:Tumor Cells, Cultured
D011508:Chondroitin Sulfate Proteoglycans; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; C121380:P3h1 protein, rat; D011509:Proteoglycans; D003871:Dermatan Sulfate; D012313:RNA; D064828:Prolyl Hydroxylases; C485657:P3H1 protein, human; D019765:Chondroitin ABC Lyase