GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates directs disease activity of ulcerative colitis.
BACKGROUND
A diffuse lymphocyte infiltrate is 1 of the characteristic features of ulcerative colitis (UC). Such lymphocyte recruitment requires lymphocyte rolling mediated by L-selectin ligand carbohydrates (6-sulfo sialyl Lewis X-capped O-glycans) and/or mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venule (HEV)-like vessels. The present study was undertaken to elucidate the role of MAdCAM-1 posttranslationally modified ("decorated") with L-selectin ligand carbohydrates in UC pathogenesis and consequent clinical outcomes.


METHODS
Biopsy specimens composed of active and remission phases of UC as well as normal colonic mucosa were immunostained for CD34, MAdCAM-1, and MECA-79, and the immunostained sections were quantitatively analyzed. Reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out to evaluate transcripts of MAdCAM-1 and N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6STs). CHO and Lec2 cells transfected with CD34 and MAdCAM-1 together with enzymes involved in L-selectin ligand carbohydrate biosynthesis were analyzed by immunofluorescence, FACS, and Western blotting to characterize the biochemical properties of GlcNAc6STs.


RESULTS
The number of MAdCAM-1(+) vessels was increased in UC, with no significant difference between active and remission phases. An increased ratio of MECA-79(+) to MAdCAM-1(+) vessels with preferential GlcNAc6ST-1 transcripts was observed in the active phase of UC compared to the remission phase. MAdCAM-1 protein was colocalized with L-selectin ligand carbohydrates at the luminal surface of HEV-like vessels in situ. GlcNAc6ST-1 preferentially utilizes MAdCAM-1 as a scaffold protein for GlcNAc-6-O-sulfation in L-selectin ligand carbohydrate biosynthesis.


CONCLUSIONS
UC disease activity is not regulated by expression of MAdCAM-1 protein itself, but rather by GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates.
Kobayashi
Motohiro
M
Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Japan. motokoba@shinshu-u.ac.jp
Hoshino
Hitomi
H
Masumoto
Junya
J
Fukushima
Mana
M
Suzawa
Kenichi
K
Kageyama
Shunsuke
S
Suzuki
Manabu
M
Ohtani
Haruo
H
Fukuda
Minoru
M
Nakayama
Jun
J
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules; D003093:Colitis, Ulcerative / Q000378:metabolism* / Q000473:pathology*; D006224:Cricetinae; D003412:Cricetulus; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism*; D019041:L-Selectin / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009088:Mucoproteins / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000080506:Sialyl Lewis X Antigen; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
D000954:Antigens, Surface; D015815:Cell Adhesion Molecules; D007136:Immunoglobulins; D019041:L-Selectin; D008024:Ligands; D008565:Membrane Proteins; D009088:Mucoproteins; D009844:Oligosaccharides; D012333:RNA, Messenger; D000080506:Sialyl Lewis X Antigen; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C069886:L-selectin counter-receptors; C504265:MADCAM1 protein, human