RESUMEN
A systematic series of chitobiose-modified pentapeptides with sequence variations of hydrophobic leucine and hydrophilic serine were synthesized. The resulting glycopeptides were used as molecular probes to elucidate aglycon peptide specificity of the glycoprotein-folding sensor enzyme UGGT. Inhibitory experiments with a synthetic fluorescent glyco-substrate and the glycopeptides revealed that UGGT prefers a serine residue directly linked to C-terminal of the N-glycosylation site in its substrate recognition.
Asunto(s)
Glucosiltransferasas/antagonistas & inhibidores , Glicopéptidos/metabolismo , Sondas Moleculares/metabolismo , Secuencia de Aminoácidos , Compuestos de Boro/química , Disacáridos/química , Glucosiltransferasas/metabolismo , Glicopéptidos/síntesis química , Glicopéptidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Sondas Moleculares/química , Unión Proteica , Especificidad por SustratoRESUMEN
BACKGROUND: Peptide: N-glycanase is a deglycosylation enzyme releasing N-glycan from glycoproteins. Although glycan specificity analysis of this enzyme has been reported, recognition requirements for the peptide sequence have not been precisely elucidated. OBJECTIVE: In this study, we carried out peptide specificity analysis of several peptide:N-glycanases. METHODS: Using synthetic chitobiose-pentapeptide substrates having a systematic series of amino acid sequences composed of hydrophobic leucine and hydrophilic serine, we examined the peptide specificities of peptide: N-glycanases comprising yeast cytoplasmic PNGase, bacterial PNGase F, and plant PNGase A by ultra-performance liquid chromatography combined with electrospray ionization mass spectrometry. RESULTS: We found that each of the PNGases had higher activity for the more hydrophobic (leucinerich) chitobiose-pentapeptides, although the sensitivities of the PNGases for hydrophobicity varied. Cytoplasmic PNGase showed broad specificity. In contrast, PNGase A showed moderate specificity. PNGase F showed the highest specificity. CONCLUSION: PNGases from different origins had similar but significantly independent peptide specificities.