Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for collagen affinity.
We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.
Kaufmann
Brigitte
B
Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, D-50931 Cologne, Germany.
Müller
Stefan
S
Hanisch
Franz-Georg
FG
Hartmann
Ursula
U
Paulsson
Mats
M
Maurer
Patrik
P
Zaucke
Frank
F
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D001842:Bone and Bones / Q000737:chemistry; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008358:Mannose / Q000737:chemistry; D051379:Mice; D015676:Osteonectin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D012513:Sarcoma, Experimental / Q000737:chemistry; D020349:Surface Plasmon Resonance
D015676:Osteonectin; D011134:Polysaccharides; D003094:Collagen; D006026:Glycoside Hydrolases; D008358:Mannose