Further purification and characterization of newly synthesized anionic glycoconjugates secreted by cultured UMR-106 cells: evidence that the major anionic glycoconjugate secreted by these cells is similar to bone sialoprotein II.
Following incubation of UMR-106 cells for 48 h in the presence of [3H]glucosamine and [35S]sulfate, the newly synthesized anionic glycoconjugates were isolated from the culture medium by cetylpyridinium chloride/ethanol precipitation and further separated by DEAE-Sephacel chromatography into two radiolabelled fractions, a major component, UM I, and a minor component, UM II. UM I appeared to be homogeneous as shown by Sepharose CL-4B chromatography under dissociative conditions, and SDS-polyacrylamide gel electrophoresis. It showed a molecular mass of approximately 93 kDa on 4-15% gels. UM I was partially degraded by brief treatment with trypsin, releasing a small, terminal peptide that contained 47.6% of 35S but no 3H. Treatment of UM I with neuraminidase and 0.1 N H2SO4 (1 h at 80 degrees C), respectively, released 27% 3H and 38.4% 3H plus 41% 35S, suggesting the presence of a significant number of sialic acid residues, as shown by Sephadex G-50 chromatography of the digests. Amino acid analysis showed that the UM I glycoconjugate was rich in acidic amino acids (12.6% aspartic acid and 21.2% glutamic acid residues) and its N-terminal sequence was Phe-Ser-Met-Lys-Asn-Phe-, which is identical to the published N-terminal amino acid sequence of rat bone sialoprotein II. Keratanase treatment of UM I released 26% of the incorporated radioactivity, suggesting the presence of keratan sulfate chains. UM II contained a chondroitinase ABC-sensitive proteoglycan.
Chopra
R K
RK
Department of Medicine, Queen's University, Kingston, Ont., Canada.
Anastassiades
T P
TP
Lohnes
D
D
Jones
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000838:Anions / Q000737:chemistry* / Q000378:metabolism; D002461:Cell Line, Transformed / Q000378:metabolism; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D006001:Glycoconjugates / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism; D012516:Osteosarcoma / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D012795:Sialoglycoproteins / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D000838:Anions; D006001:Glycoconjugates; D012795:Sialoglycoproteins; D009439:Neuraminidase; D014357:Trypsin; D002810:Chondroitinases and Chondroitin Lyases