A non-sulfated form of the HNK-1 carbohydrate is expressed in mouse kidney.
The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Gal beta1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin alpha and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate.
Tagawa
Hideki
H
Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Kizuka
Yasuhiko
Y
Ikeda
Tomoko
T
Itoh
Satsuki
S
Kawasaki
Nana
N
Kurihara
Hidetake
H
Onozato
Maristela Lika
ML
Tojo
Akihiro
A
Sakai
Tatsuo
T
Kawasaki
Toshisuke
T
Oka
Shogo
S
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D018959:CD57 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics
D018959:CD57 Antigens; D017931:DNA Primers; D012333:RNA, Messenger; D014453:Glucuronosyltransferase