Detection of 2-O-sulfated iduronate and N-acetylglucosamine units in heparan sulfate by an antibody selected against acharan sulfate (IdoA2S-GlcNAc)n.
The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of alpha-d-N-acetylglucosaminyl-2-O-sulfo-alpha-l-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V(H)3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease.
ten Dam
Gerdy B
GB
Department of Biochemistry and Pathology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, Nijmegen 6500 HB, The Netherlands.
van de Westerlo
Els M A
EM
Smetsers
Toon F C M
TF
Willemse
Marieke
M
van Muijen
Goos N P
GN
Merry
Catherine L R
CL
Gallagher
John T
JT
Kim
Yeong S
YS
van Kuppevelt
Toin H
TH
D000117:Acetylglucosamine / Q000737:chemistry*; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D016466:CHO Cells; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D006025:Glycosaminoglycans / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D007067:Iduronic Acid / Q000737:chemistry*; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D008297:Male; D008545:Melanoma / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D012908:Snails
D000117:Acetylglucosamine; D000906:Antibodies; D004187:Disaccharides; D000939:Epitopes; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D007067:Iduronic Acid; D009844:Oligosaccharides; C106771:acharan sulfate