| Asparagine-linked glycosylation(N-glycosylation)is a major covalent modification of proteins in eukaryotes and plays an indispensable role in the protein quality control,sub-localization and information flow.Oligosaccharyltransferase(OST)functions on the core step in N-glycosylation,which is responsible to transfer the preassembled N-glycans(Glc3Man9NAc2)to the asparagine residues in conserved motif(Asn-X-Ser/Thr,X represents any amino acid except proline)on the nascent peptide in the endoplasmic reticulum.OST is a multi-subunit complex enzyme and the catalytic subunit STT3 differentiates into two subtypes,STT3 a and STT3 b in plant and animal.While defects in STT3 a Arabidopsis thaliana result in unfolded protein response(UPR)and aberrant responses against stress.the mutations in isogene STT3 b cause no obvious phenotype.Simultaneous disruption of both catalytic subunits is embryo lethal in Arabidopsis,indicating that STT3 a and STT3 b function redundantly but also distinctly.To unravel the underlying mechanisms of two OST isoforms function distinctly in plant,STT3 a and STT3 b were analyzed from evolution,physiology,biochemistry,structure comparison.The main findings are listed below:(1)Phylogenetic analysis showed that the separation of two STT3 genes in plants can be traced back to the common ancestor of green plants,indicating the differentiation of STT3 a and STT3 b occurs independently in plants.Consistent with this,STT3 a and STT3 b have their own specific conserved gene structures.In addition,compared to STT3 b,the STT3 a gene promoter has more cis-regulatory elements to stress response.(2)STT3a showed higher expression level than STT3 b in yound Arabidopsis seedlings and there was no transcriptional compensation between STT3 a and STT3 b genes under salt stress.In addition,STT3 b could not restore the stt3a-1 mutant phenotype either driven by its own promoter or by STT3 a promoter.(3)Through the genetic hybridization of sugar chain synthesis mutants(alg3,ebs3 and ebs4)with stt3 a and stt3 b,the catalytic and transfer ability of OST on different sugar chains were analyzed.Under the same growth conditions,the double mutants of stt3 b ebs were more WT-looking than stt3 a ebs.Moreover,the stability of glycoprotein RSW2 was closely related to the integrity of N-glycan.The results support that OST with STT3 a and STT3 b as catalytic subunits can transfer complete and incomplete Nglycans,respectively,but STT3 a has higher catalytic ability than STT3 b.(4)Through sequence alignment,structural simulation and region substitution,it was found that the chimeric proteins consisting of intermediate region(371-540aa)or the C-terminal spherical region(541-779aa)of STT3 a with STT3 b could partially restore the salt-sensitive phenotype of stt3a-2 and the glycosylation status of PDI,suggesting that these two regions are involved in the specific functions of STT3 a.(5)Mass spectrometry analysis(LC-MS)showed that the subunits associated with STT3 a and STT3 b were different,which was further validated by Bimolecular Fluorescence Complementation(Bi FC).Besides of common assistant subunits(HAP6,DGL1,DC2-like and KCP2),STT3 a also cooperated with OST1 and OST3/6 subunits.(6)By comparing the occupancy of glycosylation sites in the wild-type and stt3a-2mutant by proteomics analysis,it was found that OST with STT3 a as catalytic subunit could modify the second site of adjacent glycosylation sites.In conclusion,the molecular mechanism of the functional differences between STT3 a and STT3 b,two subtypes of OST catalytic subunit STT3,was studied from expression level,sugar chain transfer ability,glycosylation status of substrate protein,functional difference regions and auxiliary subunits.The results showed that STT3 a and STT3 b might exist differences on ability to transfer sugar chains and preference on glycosylation sites.The functional differences between two proteins may be due to the recruitment of different auxiliary subunits. |
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