After O-mannosylated dystroglycan is transported from the ER to t

After O-mannosylated dystroglycan is transported from the ER to the Golgi apparatus, and then POMGnT1 … Individual PMTs have different specificities for protein substrates (10), suggesting the presence of some sequence for recognition by PMTs, but the sequence

has not been identified. On the other hand, in mammals, O-mannosylated proteins are rare. In mammals, O-mannosylation may require Inhibitors,research,lifescience,medical a specific sequence because we detected O-mannosyltransferase activity when a GST-fused mucin-like domain of α-dystroglycan (amino acid residues 316-489) was used as an acceptor (14). To address the regulation of O-mannosylation, we tried to determine whether substrates require a specific amino acid sequence to be recognized by O-mannosyltransferases. Inhibitors,research,lifescience,medical To answer this question, we synthesized a series of peptides that fully covered the mucin-like domain of α-dystroglycan and we examined whether these peptides worked as Epigenetic inhibitor chemical structure acceptors for protein O-mannosylation. The results showed that two similar peptide sequences, corresponding to residues 401-420 (IRPTMTIPGYVEPTAVATP) and 336-355 (SRIVPTPTSPAIAPPTETM), respectively, were strongly mannosylated, while other peptides from α-dystroglycan and peptides of various mucin tandem-repeat regions were poorly mannosylated (17). Peptides 401-420 and 336-355 contained four and Inhibitors,research,lifescience,medical six Ser/Thr residues, respectively.

Substitution of Ala residues for the Ser or Thr residues showed that Thr414 of peptide 401-420

and Thr351 of peptide 336-355 were prominently modified by O-mannosylation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Edman degradation analysis of the mannosylated peptide 401-420 indicated that Thr414 Inhibitors,research,lifescience,medical was the Thr residue that was most prominently modified by O-mannosylation Inhibitors,research,lifescience,medical and that O-mannosylation occurred sequentially rather than at random. Based on these results, we proposed a preferred amino acid sequence (IXPT(P/X)TXPXXXXPTX(T/X)XX) for mammalian O-mannosylation. A BLAST search for proteins with this sequence turned up only α-dystroglycan, suggesting that the primal O-mannosylated protein is α-dystroglycan (17). In contrast, O-mannosylation may occur by a different mechanism in S. cerevisiae. Recently, Hutzler et al. reported (18) that Pmt4p mediates Tolmetin O-mannosylation of Ser/Thr-rich membrane-bound proteins, whereas Pmt1/Pmt2 complexes act on both, soluble and membrane-bound proteins. O-Mannosyltransferase activity does not depend on the membrane-anchoring sequence, as long as it is flanked by a Ser/Thr-rich domain facing the ER lumen. In contrast to human O-mannosylation signals, Pmt4 O-mannosylation signals are not just linear sequences of proteins. Thus, it is possible that mammalian O-mannosylation requires a specific amino acid sequence while yeast O-mannosylation does not.

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