Significance of sugar moiery of recombinant glycoproteins

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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