An important open question of the study herein is whether inhibition of other glycosylation events occurring in the Golgi apparatus such as inhibition of transporters for UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, or Golgi apparatus glycosyltransferases lead to inhibition of nonglycoprotein synthesis, induction of ER stress, and inhibition of protein translation. A similar question pertains to inhibition of transport into the Golgi apparatus lumen by transporters for adenosine 3′-phosphate 5′-phosphosulfate as well as ATP. Although such studies will be easier to conduct in mammalian cell culture such as HeLa cells, extension of them to other eukaryotic cell culture systems and to multicellular organisms will be important.
Bovine, murine and porcine -lactalbumin genes have all been sequenced and their proteins have molecular weights of about 14 kD. -Lactalbumin is produced at a concentration of approximately 0.2 to 1.8 mg/ml in the milk of most mammals. It is synthesized in the rough endoplasmic reticulum and passes to the Golgi complex where it interacts with GT. In the mammary Golgi apparatus, -LA combines with G and alters the substrate specificity of galactosyltransferase from -acetylglucosamine (GlcNAc) to glucose. This modified complex transfers galactose to glucose rather than to -acetylglucosamine.
THE ROLE OF THE GOLGI APPARATUS IN THE …
The GAG polymerizing and modifying enzymes are thought to act in a coordinated fashion,facilitating concomitant synthesis and sulfation of GAGs. In vitro incubation ofGolgi-enriched subcellular fractions after chlorate treatment of MDCK cells revealed noincrease in sulfate incorporation (K. Prydz, unpublished observation). This suggests thatpost-synthesis sulfation of undersulfated GAG chains does not occur. However, the degree ofco-localization and cooperation between the enzymes involved is uncertain. Oligomers of CSsynthases 1 to 3 facilitate efficient synthesis of CS GAGs (; ). Pull-down experiments, aswell as morphological data, demonstrate enzyme complex formation in the Golgi apparatus,with functional consequences for CS synthesis (; ). A differential combination of CSsynthesizing enzymes is a potential regulatory mechanism of GAG synthesis, but thedistribution of these complexes within the Golgi apparatus and their relation to Golgiorganization and transport have not been resolved. For instance, CS 4-O-sulfation of decorinreappear more rapidly than 6-O-sulfation after reversal of BFA treatment in rat fibroblasts(). Enzymesinvolved in HS GAG synthesis and modification, EXT1 and 2 (), EXTL 3 (), and NDSTs (; ), oligomerize and form multienzymecomplexes in the Golgi apparatus. This “gagosome” model suggests that competition betweenvarious enzymes for participation in functional complexes could regulate chain length andsulfation intensity of HS GAGs.
Glycoproteins: Synthesis and Clinical Consequences
In addition to the synthesizing enzymes, availability of precursors influences GAGsynthesis. UDP-sugars and PAPS are synthesized in the cytoplasm and translocated into theGolgi lumen by nucleotide sugar transporters (NSTs) and PAPS transporters (PAPSTs).Translocation into the restricted Golgi luminal space increases the concentration 50- to100-fold (). The NSTs harboring translocation activities required for PG synthesis haveall been cloned () and display variable substrate specificity (). Homodimer formation has beenreported for the GDP-mannose transporter in yeast (). Also, interaction between aUDP-gal transporter and a galactosyltransferase has been described (). However, to what extentpartitioning and distribution of NSTs and PAPSTs within the Golgi complex contribute tovariability in PG structure is not known. We have investigated the role of PAPST1 in thesynthesis of GAGs in polarized MDCK cells (). By stable expression ofrecombinant PAPST1-GFP in MDCK cells, CS sulfation in the apical pathway was increased. Whenisolating Golgi vesicles from these cells by subcellular fractionation, we could observeincreased PAPS uptake and GAG synthesis in vitro (). The effect on CS sulfation couldbe explained by elevated PAPS concentration in the Golgi lumen. However, the differentialeffect on CS sulfation in the apical and basolateral pathways suggests pathway-specificchanges. shows the locationof PAPST1-GFP at one side of the Golgi, presumably in the TGN. Further investigations intopossible domain formations, involving the NSTs and PAPSTs, together with the synthesizingenzymes (already suggested to operate in complexes), are needed to clarify the effects ofpossible co-localization and/or complex formation.
O-linked glycoproteins: The synthesis of O ..
Enzymes involved in modification of the GAG chains, the epimerases and sulfotransferases(reviewed in ), have been observed in oligomeric complexes. The HS-modifying C-5-epimerase,which converts glucuronic acid to iduronic acid, and the 2-O-sulfotransferase do interact(). The6-O-sulfotransferase involved in HS sulfation forms oligomers required for Golgilocalization and enzyme activity (). In MDCK cells, we have observed a tendency for there to be more6-O-sulfated CS in the basolateral pathway (; ). This could be due to partitioningof enzymes in the Golgi apparatus into different synthesizing domains or pathways, asdepicted in and discussed in.