Heterogeneity in glycosylation of dermatan sulfate proteoglycan core proteins isolated from human aorta.
Small proteoglycans were dissociatively extracted from normal human thoracic aorta with 4 M guanidine hydrochloride containing protease inhibitors, and purified by Sepharose CL-4B chromatography, dissociative cesium chloride density gradient centrifugation, and diethylaminoethyl cellulose chromatography. The intact proteoglycans migrated in the 270,000-340,000 range on 4-20% sodium dodecyl sulfate polyacrylamide gradient gels. Core proteins prepared following digestion of the intact proteoglycan monomer with chondroitinase ABC consisted of a major Coomassie blue-staining protein band of 50,000 along with a minor band of 44,000. Subsequent studies using endoglycosidases H, F, and N-glycanase demonstrated that mainly complex type N-linked glycans were present on the 50,000 cores while the 44,000 cores appeared to be devoid of N-linked glycans. Western blotting demonstrated that both of these cores were recognized by the monoclonal antibody 2-B-6, indicating the presence of the terminal 4-sulfated unsaturated disaccharide (delta Di-4S) remaining on the linkage region following chondroitinase ABC digestion. In contrast, a diffuse pattern of delta Di-4S epitopes ranging from 50,000 to approximately 60,000 was observed following chondroitinase AC II digestion of the dermatan sulfate proteoglycan, suggesting the presence of iduronate residues in close proximity to the glycosaminoglycan-linkage region. Conversely, the large chondroitin sulfate-proteoglycan core proteins from aorta (Mr 200,000-400,000) did not react with either monoclonal antibody 3-B-3 (recognizing the terminal delta DI-6S) or 2-B-6 following chondroitinase AC II digestion, although both delta DI-4S and delta DI-6S were present on these cores following chondroitinase ABC digestion. Additional studies using antisera against synthetic peptides derived from sequences of the core proteins of human bone small PG I and PG II indicated the presence of both gene products in PG isolated from human thoracic aorta. The Mr approximately 44,000 and 50,000 core proteins represent small PG I type cores while a closely spaced doublet (Mr 49,000 and 51,000) represented small PG II type cores. The results demonstrate that the core proteins of dermatan sulfate proteoglycan from human aorta are heterogeneous in primary structure and in the content of N-linked glycans.
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Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103.
Wagner
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D000118:Acetylglucosaminidase / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D053674:Aggrecans; D000581:Amidohydrolases / Q000378:metabolism; D001013:Aorta, Thoracic / Q000737:chemistry*; D002808:Chondroitin Lyases / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D037181:Lectins, C-Type; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008875:Middle Aged; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011509:Proteoglycans
D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D002808:Chondroitin Lyases