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RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature
Author(s) -
HosonoFukao Tomomi,
OhtakeNiimi Shiori,
Nishitsuji Kazuchika,
Hossain Md. Motarab,
van Kuppevelt Toin H.,
Michikawa Makoto,
Uchimura Kenji
Publication year - 2011
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.22690
Subject(s) - epitope , disaccharide , heparan sulfate , chemistry , glycosaminoglycan , immunoelectron microscopy , microbiology and biotechnology , biochemistry , immunohistochemistry , blot , basement membrane , biology , antibody , immunology , gene
Abstract RB4CD12 is a phage display antibody that recognizes a heparan sulfate (HS) glycosaminoglycan epitope. The epitope structure is proposed to contain a trisulfated disaccharide, [–IdoA(2‐OSO 3 )‐GlcNSO 3 (6‐OSO 3 )–], which supports HS binding to various macromolecules such as growth factors and cytokines in central nervous tissues. Chemically modified heparins that lack the trisulfated disaccharides failed to inhibit the RB4CD12 recognition of HS chains. To determine the localization of the RB4CD12 anti‐HS epitope in the brain, we performed an immunohistochemical analysis for cryocut sections of mouse brain. The RB4CD12 staining signals were colocalized with laminin and were detected abundantly in the vascular basement membrane. Bacterial heparinases eliminated the RB4CD12 staining signals. The RB4CD12 epitope localization was confirmed by immunoelectron microscopy. Western blotting analysis revealed that the size of a major RB4CD12‐positive molecule is ∼460 kDa in a vessel‐enriched fraction of the mouse brain. Disaccharide analysis with reversed‐phase ion‐pair HPLC showed that [–IdoA(2‐OSO 3 )‐GlcNSO 3 (6‐OSO 3 )–] trisulfated disaccharide residues are present in HS purified from the vessel‐enriched brain fraction. These results indicated that the RB4CD12 anti‐HS epitope exists in large quantities in the brain vascular basement membrane. © 2011 Wiley‐Liss, Inc.