Significance of sugar moiery of recombinant glycoproteins

γ-谷氨酰转肽酶是一种位于细胞膜外表面的异源二聚体糖蛋白，在谷胱甘肽代谢过程中起着重要作用。它催化谷氨酰基部分从谷胱甘肽和相关化合物转移到各种氨基酸和二肽。我们建立了γ-谷氨酰转肽酶在空泡病毒/昆虫系统中的表达系统，并对该酶的活性部位进行了分析。我们发现，该酶活性所需的丝氨酸残基是通过对保守的丝氨酸残基进行定点突变而确定的，该突变是根据人、大鼠、猪和两种细菌酶的光亚基所需的比对来确定的。用杆状病毒-昆虫细胞系统表达了用丙氨酸取代这些丝氨酸残基的重组人酶。这些结果表明，Ser-45T1和Ser-452都位于能够与γ-谷氨酰基相互作用并参与催化的位置，可能是…更多的是作为亲核剂或通过稳定过渡态和残留物在各种动植物物种中被发现保存得很好。这些工作是与纽约康奈尔大学医学院的Alton Meister博士和Mary Anderson博士合作进行的，我们在J.Biol上发表了几篇论文。载脂蛋白E(ApoE)在糖尿病等高血糖条件下发生非酶糖基化，导致与肝素的总结合力和亲和力降低。在正常和糖尿病条件下，血浆中的载脂蛋白E在极低密度脂蛋白组分中也会发生糖基化。然而，在糖尿病患者中，糖基化作用显著增加，最高可达2-3倍。此外，载脂蛋白E刺激肝素结合的EGF样生长因子(HB-EGF)的活性，HB-EGF是血管平滑肌细胞的有丝分裂原。提示载脂蛋白E和HB-EGF可能在糖尿病动脉粥样硬化的发病机制中起重要作用。这些工作是与法国南希的Gerard Siest、Maria Wellman和Vladimir Shuvaev博士合作完成的。较少

gamma-Glutamyl transpeptidase, a heterodimeric glycoprotein anchored to the extra cellular surface of cell membrane, plays an important role in glutathione metabolism. It catalyzes the tansfer reaction of a gamma-glutamyl moiety from glutathione and related compounds to variety of amino acids and dipeptides. We have established tha expression system for gamma-glutamyl transpeptidase in vaculovirus/insect system and analyzed the active sites of the enzyme. We found the serine residues required for batalysis of the enzyme was identified by site-specific mutagenesis of the conserved serine residues on the basis of required alignment of the light subbing of human, rat pig and two bacterial enzymes. Recombinant human enzyme with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. There results suggest that both Ser-45tl and Ser-452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably … More as nucleophiles or through stabilization of the transition state and the residues ware found to be well conserved in various animal and plant species. These works have been carried out in collaboration with Dr.Alton meister and Dr.Mary Anderson at the Cornell University Medical School in New York and we have published several papers in J.Biol. Chem and Proc.Natl.Acad.Sci.USA.Apolipoprotein E (ApoE) undergoes non-enzymatic glycosylation (glycation) under hyperglycemic conditions such as diabetes and results in the decrease of total binding and affinity to heparin. In plasma, ApoE also undergoes glycation in the very low density lipoprotein fractions under normal and diabetic conditions. In diabetic patients however, glycation is dramatically increased up to 2-3 folds. Moreover, ApoE stimulates the activity of heparin binding EGF like growth factor (HB-EGF) which is a mitogen to vascular smooth muscle cells. These results indicate that both apo E and HB-EGF may play a critical role in the pathogenesis of atherogenic changes in the diabetes. These works have been done in collaboration with Drs.Gerard Siest, Maria Wellman and Vladimir Shuvaev at Nancy, France. Less