Processing enzyme glucosidase II: proposed catalytic residues and developmental regulation during the ontogeny of the mouse mammary gland.
Following the action of glucosidase I to clip the terminal alpha1,2-linked glucose, glucosidase II sequentially cleaves the two inner alpha1,3-linked glucose residues from the Glcalpha1,2Glcalpha1,3Glcalpha1,3Man(9)GlcNAc(2) oligosaccharide of the incipient glycoprotein as it undergoes folding and maturation. Glucosidase II belongs to family 31 glycosidases. These enzymes act by the acid-base catalytic mechanism. The cDNA of the wild-type and several mutant forms of the fusion protein of the enzyme in which mutations were introduced in the conserved motif D(564)MNE(567) were expressed in Sf9 cells, and the proteins were purified on Ni-NTA matrix. The catalytic activity of the purified proteins was determined with radioactive Glc(2)Man(9)GlcNAc(2) substrate. The results show that the aspartate and glutamate within the D(564)MNE(567) motif can serve for catalysis, most likely as the acid-base pair within the active site of the enzyme. The developmental regulation of glucosidase II was studied during the ontogeny of the mouse mammary gland for its growth and differentiation. The mRNA of both alpha and beta subunits of the enzyme, immunoreactive alpha and beta subunits, and enzyme activity were measured over the complete developmental cycle. The changes in all the parameters were consistent with similar fluctuations with several other enzymes of the N-glycosylation machinery reported earlier, reaching a three- to fourfold increase over the basal level in the virgin gland at the peak of lactation. Altogether it appears that there is a coordinated regulation of the enzymes involved in protein N-glycosylation during the development of the mouse mammary gland.
Feng
Jie
J
Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA.
Romaniouk
Andrew V
AV
Samal
Siba K
SK
Vijay
Inder K
IK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008321:Mammary Glands, Animal / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D006023:Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases