小鼠睪丸內Transglutaminase受質的純化與鑑定

Abstract

TGase（transglutaminase）最早是由Heinrich Waelsch於1957年所發現，是一個在有鈣離子存在的情形下，能對蛋白質的Gln residue上γ-carboxamide group與Lys residue上ε-amino group進行交聯作用（cross-linking）的一個酵素。目前有九種的TGase家族基因已被證實，而其中有八種都可表現為具有活性的酵素。 睪丸是男性的生殖和內分泌系統的器官之一，主要的功能是提供精蟲形成（spermatogenesis）和分泌男性賀爾蒙，像是大家較為熟悉的睪丸素（testosterone）。一般精蟲形成所需的時間約為70天，從青少年時期開始，一直到死，雖然隨著年齡的增長，spermatogenesis的情形會略為下降，但還是持續的進行中。 在本實驗中，我們利用小鼠的睪丸為材料，以蛋白質體學的方法去純化以及鑑定小鼠睪丸內TGase的受質，希望在一個像睪丸這樣持續進行細胞生長與分化的組織裡，了解TGase所扮演的角色為何。經由純化及MS/MS鑑定過後，我們發現了數十種蛋白質在小鼠睪丸內可能為TGase的受質，在經由重組TGase受質蛋白的體外 transamidation反應以及TGase 受質 pull-down的immunoblotting assay，我們也證實了這些蛋白在體外的確為TGase的受質。 未來我們希望從中選取了幾個可能為TGase受質的蛋白，觀察其TGase進行transamidation過後，會對這些蛋白的酵素活性、蛋白與蛋白之間的作用力或是protein translocation會有怎樣的影響，進而能更加了解TGase在睪丸內的角色與功能。

TGase (Transglutaminase) was first discovered by Heinrich Waelsch in 1957. It is a calcium-dependent enzyme, which catalyzes the formation of isopeptide linkages between the γ-carboxamide group of a glutamine residue and the ε-amino group of a lysine residue. Some physiological roles are demonstrated for TGase, including blood coagulation, regulation of cell growth and differentiation, and programmed cell death. Testis is a component of both the reproductive system and the endocrine system. The respective functions of the testis are: producing sperm (spermatozoa) and producing male sex hormones, of which testosterone is the best known . The process of spermatogenesis takes approximately 70 days, it starts at puberty and usually continues uninterruptedly until death, although a slight decrease in the quantity of produced sperm can be discerned with increase in age. In this research, we adopted proteomics methods to purify and identify TGase substrates. After MS/MS analysis, we have identified over 70 potential TGase substrates, some of them have been verified in our lab by in vitro transamidation of recombinant TGase substrates and/or by immunoblotting of TGase substrates in a pull-down assay. More importantly, most of the identified TGase substrates are novel and have not yet been studied. Our future work will be to examine the effects of transamidation on the feature of each novel substrate, in terms of enzyme activity, protein-protein interaction, and protein translocation.