The Functional Research Of The Polypeptide N-Acetylgalactosaminyltransferase 18 In Endoplasmic Reticulum

Mucin-type O-glycosylation,also known as O-Gal NAc glycosylation,is among the most common and fundamental post-translational protein modifications and plays critical roles in various physiological and pathological processes.Thus,investigation of the regulation mechanism of O-Gal NAc glycosylation is of great significance for understanding the roles of O-glycans and O-glycoproteins in biological events.In human,mucin-type O-glycosylation is initiated by a family of 20 conserved UDP-Gal NAc:polypeptide N-acetylgalactosaminyltransferases(pp Gal NAc-Ts),which catalyzes the transfer of the N-acetylgalactosamine(Gal NAc)to the hydroxyl group of serine or threonine.These pp Gal NAc-T isoforms,notably,display tissue-specific expression and substrate-specific preference in adult mammals,which is the key to the diversity and complexity of mucin-type O-glycans.Previously,we first discovered that pp Gal NAc-T18 is a new member of pp Gal NAc-T that is only existed in vertebrates and is predominantly distributed on the endoplasmic reticulum(ER).Interestingly,although it exhibits no classical catalytic activity in vitro,it can affect the O-glycosylation and cell growth in vivo.These results imply the important roles of pp Gal NAc-T18 in biological processes,which is obviously distinct from others pp Gal NAc-Ts localized to the Golgi apparatus.In this study,we performed a systematic functional analysis of pp Gal NAc-T18 in the ER using various biochemical and biological approaches.Using both in vitro and in vivo models,we found that pp Gal NAc-T18 is important for inflammation-induced ER stress.The main results are as follows:1.The relationship between the expression of pp Gal NAc-T18 and ER stress in vitro.Silencing of pp Gal NAc-T18 in cells decreased O-glycosylation levels and activated ER stress leading to apoptosis.After treatment with chemical chaperone 4-phenylbutyric acid(PBA)or forced expression of pp Gal NAc-T18 in the pp Gal NAc-T18 knockdown cell,these defects could be significantly alleviated,suggesting that pp Gal NAc-T18exerts its functions in ER homeostasis and protein O-glycosylation in a chaperone-like manner.Furthermore,we found both of the wildtype and the Mn²⁺binding site mutant pp Gal NAc-T18 could rescue the O-glycosylation and alleviated the ER stress in pp Gal NAc-T18 knockdown cells.These results reveal a novel molecular role of pp Gal NAc-Ts that the ER-localized pp Gal NAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner,and provide a new insight into the molecular mechanisms underlying pp Gal NAc-T-mediated O-glycosylation and ER homeostasis.2.The function of pp Gal NAc-T18 on ER stress in a mouse neuroinflammation model.To further confirm the biological function of pp Gal NAc-T18 on ER stress in vivo,we constructed a mouse neuroinflammation model induced by lipopolysaccharide(LPS)and a neuro-specific pp Gal NAc-T18 conditional knockout mouse using the Cre-loxp recombination system.We found that concomitant with enhanced expression of ER stress-associated proteins,pp Gal NAc-T18 was specifically induced in neurons of the mouse cerebral cortex during LPS-induced inflammation.It is noteworthy that the expression of ER stress-associated proteins was clearly upregulated in c T18^fl/fl mice either before or after the injection of LPS as compared to the c T18^wt/wt mice.These results indicate pp Gal NAc-T18 plays an important role in the regulation of inflammation-induced ER stress.Furthermore,the upregulation of pp Gal NAc-T18 during neuroinflammation was further verified in neuron-like PC12 cells treated with proinflammatory stimuli including TNF-?and H₂O_2.In addition,the overexpression of pp Gal NAc-T18 could significantly attenuate the ER stress and alleviate the cell apoptosis induced by neuroinflammation.These results clearly suggest the importance of ER-localized pp Gal NAc-T18 in aggravating the ER stress triggered by neuroinflammation,and provide a new insight to discover the unknown functions of glycosyltransferases.In summary,our results first demonstrate that pp Gal NAc-T18,a novel ER-localized O-Gal NAc glycosyltransferase,could regulate the O-glycosylation and ER homeostasis via a non-catalytic function,and thus plan an important role in aggravating the ER stress and cell apoptosis triggered by neuroinflammation.This study is of great significance to understand the biological function of the novel ER-localized pp Gal NAc-T in physiological and pathological processes.