Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans.
Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans.
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.
Fredrik
F
Noborn
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.
Alejandro
A
Gomez Toledo
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.
Anders
A
Green
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.
Waqas
W
Nasir
Proteomics Core Facility, Sahlgrenska Academy, University of Gothenburg, Box 413, SE-405 30, Sweden.
Carina
C
Sihlbom
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.
Jonas
J
Nilsson
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden.
Göran
G
Larson
D000818:Animals; D045744:Cell Line, Tumor; D019765:Chondroitin ABC Lyase / Q000737:chemistry*; D002809:Chondroitin Sulfates / Q000737:chemistry* / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism
D019765:Chondroitin ABC Lyase; D002809:Chondroitin Sulfates; D006497:Heparan Sulfate; D011509:Proteoglycans
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