Functional Study Of Glycosyltransferase GtB And GtC Of Cytophaga Hutchinsonii

Glycosylation modification of proteins is one of the main post-translational modifications,which is of great significance for normal function of proteins.According to different chemical bond types of protein glycosylation,we can divide into N-,O-,C-,P-,and S-glycosylation.N-glycosylation modification of Campylobacter jejuni（C.jejuni）is extensively reported.That is the whole glycosylation process mediated by pgl gene clusters,in which the oligosyltransf-erase Pg1B plays a key role.There are few reports on N-glycosylation modific ations in the phylum Bacteroidetes.Cytophaga hutchinsonii（C.hutchinsonii）,as a member of Bacteroidetes,possesses unique IX type protein secretion system（T9SS）,which participates in the secretion and anchoring of important proteins such as cellulases of C.hutchinsonii.Recently,we discovered N-glycosylation modification occurred in periplasmic space of T9SS substrate protein of C.hut chinsonii CHU₂708 conserved C-terminal domain（CTD）.But C.hutchinsonii is not similar to the pgl gene clusters of C.jejuni,which indicates that it is different from the N-glycosylation pathway of C.jejuni.As Glycosyltransferase play crucial roles in the glycosylation modification of proteins,identifying a series of glycosyltransferases involved in glycosylations are significant for revea-ling the glycosylation modification system of C.hutchinsonii.There is a 29%amino acid sequence identity between C.jejuni PglJ and CHU₀802（GtB）.GtB mutant strain is prepared and we discover that the N-gly cosylation modification of CTDCHU₂708 was weakened in the mutant,we infer that GtB participated in the N-glycosylation modification of CTDCHU₂708.At the same time,GtB also affects the anchoring of outer membrane protein such as CHU₁336（Cel9A）.Agglutinin affinity experiments have shown that GtB is also involved in glycosylation modification of some cytoplasmic proteins in C.hutchinsonii.In addition,the absence of GtB can also leads to cellulose utilization deficiency,The deficiency of transcription level of cellulase gene chu₁280 and chu₂103,the decrease of bacterial motility and the decrease of resistance to drug sensitive reagents such as dithiothreitol（DTT）and crystal violet,which indicatate that GtB makes a wide range of physiological effects.Glycosyltransferase CHU₂109（GtC）is a member of the glycosyltransferase 2 family.We found that GtC does not affect the N-glycosylation of CTDCHU₂708,the absence of GtC also leads to the failure of cellulase Cel9A anchoring in outer membrane.In addition,the absence of GtC also affects important phys-iological functions such as cellulose degration and resistance to DTT,crystal violet and other reagents.We infer that GtC may not be the key glycosyltransf-erase in the N-glycosylation process.In summary,we found GtB is an important glycosyltransferase involved in the N-glycosylation modification of T9SS substrate protein CTD.The absence of GtB can affect the glycosylation modification of various proteins,leads to defects in bacterial cellulose utilization,decreased motility,decreased resistance to DTT,crystal violet and other reagents.This provides new insight into the study of the functions of glycosyltransferases and glycosylation modifications.