Intact Golgi synthesize complex branched O-linked chains on glycoside primers: evidence for the functional continuity of seven glycosyltransferases and three sugar nucleotide transporters.
We examined the functional co-localization and continuity of glycosyltransferases and sugar nucleotide transporters in the Golgi of two Chinese hamster ovary (CHO) cell lines that synthesize different types of O-linked oligosaccharides. CHO cells normally synthesize primarily Sia2,3Galbeta1,3GalNAc- on glycoproteins. CHO cells transfected with core-2 GlcNAc transferase (Core 2) can synthesize glycoproteins containing branched O-linked oligosaccharides with poly-N-acetyllactosamines. CHO lines incubated with [(3)H]galactose and GalNAc-alpha-phenyl (GAP) as a primer, synthesize labeled glycoside products that faithfully resemble those found on the endogenous acceptors: CHO cells make Sia2,3[(3)H]Gal(beta)1,3GAP, while CHO Core2 cells synthesize GAPs with complex branched chains including poly-N-acetyllactosamines. To determine if isolated Golgi preparations make similar products, we prepared Golgi by established homogenization methods, documented their intactness, and added tracer UDP-[(3)H]Gal, unlabeled sugar nucleotides, and GAP. CHO Golgi preparations synthesized only Sia2,3[(3)H]Gal(beta)1,3GAP. CHO Core2, also made this product and a small amount of Core-2 GlcNAc transferase-dependent products. No endogenous glycoproteins were labeled. However, when either cell line was gently permeabilized with streptolysin-O or given hypo-osmotic shock, both GAP and endogenous acceptors were efficiently glycosylated within an intact functional Golgi lumen and remained there. Significantly, Golgi from CHO Core2 cells made mostly branched GAP products including some with poly-N-acetyllactosamines as complex as those made and secreted by living cells incubated with GAP. These results suggest that the lumen of the Golgi apparatus is functionally continuous or interconnected. Once glycosides diffuse into the Golgi lumen, they have access to all the sugar nucleotide transporters and glycosyltransferases used for complex GAP-based products without requiring metabolic energy or inter-vesicular transport. Glycosylation of artificial acceptors could be used to track the functional continuity or co-localization of multiple glycosyltransferases and transporters under conditions where Golgi morphology disintegrates and/or reappears.
Kim
S
S
Korea Basic Science Institute, Taejon 305-333, South Korea.
Miura
Y
Y
Etchison
J R
JR
Freeze
H H
HH
D000818:Animals; D001173:Arthrobacter / Q000201:enzymology; D001426:Bacterial Proteins; D001692:Biological Transport / Q000502:physiology; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002463:Cell Membrane Permeability / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D033704:Nucleotide Transport Proteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D009997:Osmotic Pressure; D051381:Rats; D013301:Streptolysins / Q000737:chemistry / Q000378:metabolism; D014316:Tritium / Q000737:chemistry; D014530:Uridine Diphosphate / Q000737:chemistry
D001426:Bacterial Proteins; D006027:Glycosides; D033704:Nucleotide Transport Proteins; D009844:Oligosaccharides; D013301:Streptolysins; C025964:streptolysin O; D014316:Tritium; D014530:Uridine Diphosphate; D016695:Glycosyltransferases