Use of a phage display antibody to measure the enzymatic activity of the Sulfs.
Sulf-1 and Sulf-2 are extracellular endoglucosamine 6-sulfatases, which selectively liberate the 6-O-sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact heparan sulfate (HS)/heparin chains. The Sulfs are known to regulate signaling of heparin/HS-binding protein ligands, such as morphogens and growth factors, presumably through their ability to decrease the association between the ligands and HS proteoglycans. These enzymes serve important roles in development and are dysregulated in many cancers. We previously described arylsulfatase and endoglucosamine 6-sulfatase assays for the Sulfs. RB4CD12 is a phage display anti-HS antibody. N-sulfation, 2-O-sulfation, and 6-O-sulfation are involved in its binding. In this chapter, we describe the application of RB4CD12 in ELISA, flow cytometry, and immunohistochemistry assays to measure the enzymatic activity of the Sulfs. These newly established methods should facilitate further investigation of the Sulfs in vitro and in vivo.
Uchimura
Kenji
K
Section of Pathophysiology and Neurobiology, National Center for Geriatrics and Gerontology, Aichi, Japan.
Lemjabbar-Alaoui
Hassan
H
van Kuppevelt
Toin H
TH
Rosen
Steven D
SD
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D000939:Epitopes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D019151:Peptide Library; D015238:Sulfotransferases / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000939:Epitopes; D006493:Heparin; D006497:Heparan Sulfate; D019151:Peptide Library; D015238:Sulfotransferases