| Collagen is the main component of connective tissues in animals,and comprises of 25-35% of the body’s protein content.According to the collagen structure,collagens can be separated into fibrillar and non-fibrillar groups,fibrillar being the most abundant and Type I collagen being the main fibrillar collagen,which participates in many important physiological processes,such as tissue repair and bone development.Abnormal synthesis and deposition of Type I collagen can also lead to many diseases,such as scar formation,tissue fibrosis and genetic diseases.At present,about 45% of human deaths are attributed to fibrotic diseases.Although many patients suffer from fibrotic diseases,there are no effective treatments,and the mechanism of collagen synthesis has not been fully elucidated.Therefore,exploring the mechanism of collagen synthesis and secretion at the molecular and cellular levels is of great importance for the treatment of fibrotic diseases.In the first chapter,we firstly summarized the structural characteristics and classification of collagen;secondly,we described the mechanism of Type I collagen synthesis at transcriptional level,post-transcriptional level,its post-translational modification and secretion process;thirdly,we expatiated on the structure and function of prolyl-4-hydroxylases(P4Hs),a key enzyme involved in collagen synthesis;fourthly,we introduced the mechanism of N-linked glycosylation modification in mammals;finally,We elaborated the purpose and significance of this study.In the second chapter,we elaborated the effect of P4HA1 Asn242(N242)glycosylation on the synthesis of Type I collagen.Firstly,we reported that treatment of fibroblasts for 3-6 hours with VC increased the secretion of mature collagen1 α2(col1 α2)and induced P4HA1 to produce N242 glycans.P4HA1 is an N-linked glycoprotein with two glycosylation sites,N96 and N242.After treatment with PNGaseF,we found that P4HA1 N96 glycosylation did not depend on VC,butP4HA1 N242 glycosylation depended on VC treatment and was built on N96 glycosylated P4HA1.N-linked glycosylation is one of the most common post-translational modifications,which is conducive to protein folding,stability and regulation of its function and activity.The process of N-linked glycosylation could be divided into two ways: co-translational glycosylation mediated by STT3 A complex and post-translational glycosylation mediated by STT3B/MagT1 complex.After silencing Stt3 a,Stt3b or Magt1 genes in MEF cells,we found that P4HA1 N242 glycosylation induced by VC was STT3B/MagT1 complex dependent and thus post-translational.Subsequently,we found that the P4HA1 N242 glycans was correlated with the enhanced secretion of col1 α2.To further study the correlation between N242 glycans and col 1α2,we treated Stt3 b or Magt1 silencing MEF cells with VC.Compared with control cells,P4HA1 lacked N242 glycans,pepsin resistant col 1α2 secretion was significantly reduced.Immunofluorescence staining showed that Stt3 b or Magt1 silencing decreased col1 α2 entry into Golgi apparatus after VC treatment.Co-immunocoprecipitation showed that reduced N242 glycosylation on P4HA1 decreased interaction between P4HA1 and col 1α2.Mass spectrometry also showed that P4HA1 lacking N242 glycans attenuated proline hydroxylation on col1α2,especially on proline at sites 74,272,755,773 and 1016,However,the reduction of proline hydroxylation was not universal,significantly more hydroxylated proline was detected at sites 542 and 899 on col1 α2 in Stt3 b silenced cells(Similar effects were detected in col1 α1).Therefore,N242 glycans on P4HA1 fine tune the interaction between col1 α2 and P4HA1 and affect the hydroxylation of specific proline of col1 α2.In the last chapter,we summarized the main conclusions of the above research.However,The mechanism by which P4HA1 N242 glycans alters P4HA1 binding to col1 α2 remains unknown.Meanwhile,identification of the mechanism by which the N242 glycans regulate proline hydroxylation at specific sites of collagen will require detailed structural studies. |
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