Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland.

Like animal cells, many unicellular eukaryotes modify mucin-like domains of secretory proteins with multiple O-linked glycans. Unlike animal mucin-type glycans, those of some microbial eukaryotes are initiated by ␣-linked GlcNAc rather than ␣-GalNAc. Based on sequence similarity to a recently cloned soluble polypeptide hydroxyproline GlcNAc-transferase that modifies Skp1 in the cytoplasm of the social ameba Dictyostelium, we have identified an enzyme, polypeptide ␣-N-acetylglucosaminyltransferase (pp ␣-GlcNAc-T2), that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp ␣-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp ␣-GlcNAc-T2 efficiently transfers GlcNAc from UDP-GlcNAc to synthetic peptides corresponding to mucin-like domains in two proteins that traverse the secretory pathway. pp ␣-GlcNAc-T2 is required for addition of GlcNAc to peptides in cell extracts and to the proteins in vivo. Mass spectrometry and Edman degradation analyses show that pp ␣-GlcNAc-T2 attaches GlcNAc in ␣-linkage to the Thr residues of all the synthetic mucin repeats. pp ␣-GlcNAc-T2 is encoded by the previously described modB locus defined by chemical mutagenesis, based on sequence analysis and complementation studies. This finding establishes that the many phenotypes of modB mutants, including a permeability defect in the spore coat, can now be ascribed to defects in mucin-type O-glycosylation. A comparison of the sequences of pp ␣-GlcNAc-T2 and the animal pp ␣-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action.

Section: Figsupporting

confidence: 67%

Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function

Wang

Metcalf

Wel

et al. 2003

“…We have observed that newly synthesized CD8-K and CD8-E19, in contrast to CD8-S and unmodified CD8, require several hours to undergo the initial O-glycosylation events. There is compelling evidence that these events occur in the cis/medial Golgi compartments (Roth et al, 1994;Piller et al, 1989;Verde et al, 1995;Vassard et al, 1995). This finding may thus support the view of efficient retrieval occurring upstream of these compartments.…”

Section: Discussionmentioning

confidence: 99%

Different Fate of a Single Reporter Protein Containing KDEL or KKXX Targeting Signals Stably Expressed in Mammalian Cells

Martire

Mottola

Pascale

et al. 1996

In mammalian cells, resident luminal and type I transmembrane proteins of the endoplasmic reticulum usually contain KDEL and KKXX at the carboxyl terminus. These sequences induce retrieval from compartments located downstream in the secretory pathway. It has been suggested that the retrieval may occur from multiple sites, ranging from the intermediate compartment to the trans-Golgi network. To compare the retrieval of luminal and type I membrane proteins, we have used different forms of a single reporter, the human CD8 glycoprotein, stably expressed in FRT cells. Metabolic labeling and oligosaccharide analysis show that the mechanism based on the KDEL signal is leaky. With time, the KDEL-containing CD8 form reaches the trans/ trans-Golgi network compartments, where the protein is terminally glycosylated. At this stage, the retrieval mechanism stops being effective and the protein is consequently secreted. Conversely, the mechanism based on the KKXX signal guarantees that most of the KKXXcontaining CD8 form resides in the endoplasmic reticulum, little in the Golgi complex and undetectable levels at the plasma membrane. The O-glycosylation of this protein comprises for the vast majority the sole addition of peptide-bound GalNAc that occurs in an early Golgi compartment.

“…The polypeptide GalNAc-transferases are distributed throughout the Golgi cisternae, but there is evidence for some differences in distribution among isoforms (33,34). The Oglycan processing step, involving initiation, elongation, branching, and termination of oligosaccharide chains, is believed to occur throughout the Golgi stacks and in the trans-Golgi network (for review, see Ref.…”

Section: Discussionmentioning

confidence: 99%

The Lectin Domain of UDP-N-acetyl-d-galactosamine:PolypeptideN-acetylgalactosaminyltransferase-T4 Directs Its Glycopeptide Specificities

Hassan¹,

Reis

Bennett³

et al. 2000

153

137

The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Differences in kinetic properties, substrate specificities, and expression patterns of these isoenzymes provide for differential regulation of O-glycan attachment sites and density. Recently, it has emerged that some GalNAc-transferase isoforms in vitro selectively function with partially GalNAc O-glycosylated acceptor peptides rather than with the corresponding unglycosylated peptides. O-Glycan attachment to selected sites, most notably two sites in the MUC1 tandem repeat, is entirely dependent on the glycosylation-dependent function of GalNAc-T4. Here we present data that a putative lectin domain found in the C terminus of GalNAc-T4 functions as a GalNAc lectin and confers its glycopeptide specificity. A single amino acid substitution in the lectin domain of a secreted form of GalNAc-T4 selectively blocked GalNAc-glycopeptide activity, while the general activity to peptides exerted by this enzyme was unaffected. Furthermore, the GalNAc-glycopeptide activity of wild-type secreted GalNAc-T4 was selectively inhibited by free GalNAc, while the activity with peptides was unaffected.