A specific detection of GlcNAcbeta1-6Manalpha1 branches in N-linked glycoproteins based on the specificity of N-acetylglucosaminyltransferase VI.
Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface-in particular, the production of GlcNAcbeta1-6Manalpha1 branches in N-linked glycoproteins. To identify the target glycoproteins, we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled uridine (5'-)diphosphate-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAcbeta1-6Manalpha1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto alpha1-acid glycoprotein (AGP), which is known to contain GlcNAcbeta1-6Manalpha1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcbeta1-6Manalpha1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and beta-galactosidase could be detected using the assay. One was identified as AGP from its mobility on SDS-PAGE, demonstrating the potential of this assay even with crude materials. Furthermore, this method could detect a protein that was also positively stained with leukoagglutinating phytohemagglutinin (L(4)-PHA) using glycoproteins prepared from WiDr human colon cancer cells. This method should provide a useful complement to the current method, which relies on the specificity of L(4)-PHA.
Watanabe
Tae
T
Department of Biochemistry, Osaka University Graduate School of Medicine 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Ihara
Hideyuki
H
Miyoshi
Eiji
E
Honke
Koichi
K
Taniguchi
Naoyuki
N
Taguchi
Tomohiko
T
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism*; D001681:Biological Assay; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000737:chemistry / Q000473:pathology; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008546:Melanoma, Experimental / Q000737:chemistry / Q000473:pathology; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009439:Neuraminidase / Q000494:pharmacology; D010059:Ovomucin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000494:pharmacology
D001212:Asialoglycoproteins; D006023:Glycoproteins; D011994:Recombinant Proteins; C037370:asialoovomucoid; D010059:Ovomucin; D017351:N-Acetylglucosaminyltransferases; C417184:UDP-N-acetylglucosamine N-acetyl-D-glucosaminyl-1-6-(N-acetylglucosaminyl-1-2)mannopyranosyl-1-R(N-acetylglucosamine to mannose)-1,4N-acetylglucosaminyltransferase VI; D009439:Neuraminidase; D001616:beta-Galactosidase