Glycosphingolipid-binding specificity of the mannose-binding protein from human sera.
Mannose-binding protein was purified from human serum to apparent homogeneity by affinity chromatography on mannose-Sepharose, followed by affinity chromatography on underivatized Sepharose. Approximately 0.4 mg protein was obtained from 1 liter serum. The glycosphingolipid-binding specificity of the purified protein was examined by chromatogram overlay and solid phase assays. It binds with high affinity to Lc-3Cer (GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide) and n-Lc5Cer (GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide). It does not bind to many other glycosphingolipids without terminal N-acetylglucosamine residues that were tested. Thus, these data suggest that N-acetylglucosamine-terminated glycosphingolipids may serve as cell-surface attachment sites for mannose-binding protein in vivo. In addition, the binding specificity of the protein can be used as a sensitive probe for determining the levels of Lc3Cer and nLc5Cer in tissues, as it exhibits half-maximal binding to about 10 pmol of these lipids in solid phase assays, and detects less than 20 pmol of Lc3Cer in chromatogram overlay assays. This technique was utilized to demonstrate that one sample of chronic myeloid leukemia cells contains both Lc3Cer and nLc5Cer.
Kyogashima
M
M
Laboratory of Structural Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Krivan
H C
HC
Schweinle
J E
JE
Ginsburg
V
V
Holt
G D
GD
D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D008358:Mannose / Q000378:metabolism; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D011485:Protein Binding
D002352:Carrier Proteins; D006028:Glycosphingolipids; D037241:Mannose-Binding Lectins; D008358:Mannose