Oligosaccharide sequence of human breast cancer cell heparan sulfate with high affinity for laminin.
Laminin-1 is a basement membrane glycoprotein implicated in tumor-host adhesion, which involves the cell-binding domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are necessary for binding to laminin-1 have not been characterized. To identify this laminin-binding oligosaccharide sequence, GlcNSO4-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) residues were isolated from human breast cancer cell (MCF-7)-derived HS through hydrazinolysis/high pH (4.0) nitrous acid treatment/[3H]NaBH4 reduction. These oligosaccharides were chromatographed on a laminin-1 affinity column. A high affinity dodecasaccharide was isolated and characterized. Disaccharide analysis yielded IdoA(2-SO4) --> AManR(6-SO4) as the only disaccharide upon treatment of this dodecasaccharide with nitrous acid at low pH (1.5). The sequence of laminin-binding high affinity oligosaccharide is therefore [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5[IdoA(2-SO4) --> AManR(6-SO4)]. Low affinity dodecasaccharides composed of [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5, [IdoA(2-SO4) --> GlcNSO4] were also isolated by laminin-1 affinity chromatography. Molecular modeling studies indicate that a heparin-binding peptide sequence corresponding to amino acid residues 3010-3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of laminin-1, modeled as a right-handed alpha-helix, carries an array of basic residues well placed to bind to clusters of sulfate groups on the high affinity dodecasaccharide.
Section of Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1040, USA. npartha@wfubmc.edu
N
N
Parthasarathy
L F
LF
Gotow
J D
JD
Bottoms
T E
TE
Kute
W D
WD
Wagner
B
B
Mulloy
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007797:Laminin / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D004187:Disaccharides; D006497:Heparan Sulfate; D007797:Laminin; D010446:Peptide Fragments