Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetylgalactosaminyltransferase and alpha1,3-fucosyltransferase.
Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAcbeta1-4GlcNAcbeta1-)n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Galbeta1-4GlcNAcbeta1-)n. We also stably expressed human alpha1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha1,3-fucosylated LDN (LDNF) GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-R as well as novel "poly-LDNF" structures (-3GalNAcbeta1-4(Fucalpha 1-3)GlcNAcbeta1-)n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAcbeta1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.
Kawar
Ziad S
ZS
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Haslam
Stuart M
SM
Morris
Howard R
HR
Dell
Anne
A
Cummings
Richard D
RD
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D017173:Caenorhabditis elegans; D002236:Carbohydrate Conformation; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry* / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000737:chemistry*; D005647:Fucosyltransferases / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000502:physiology*; D013058:Mass Spectrometry; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D016366:Open Reading Frames; D011134:Polysaccharides / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004187:Disaccharides; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D005643:Fucose; D005647:Fucosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C031692:galactoside 3-fucosyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; D007785:Lactose