Glycosylation of eukaryotic peptide chain initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) and its possible role in the inhibition of eIF-2 kinase-catalyzed phosphorylation of the eIF-2 alpha-subunit.
We have reported previously that a 67-kDa polypeptide (p67) present in reticulocyte lysates protects the alpha-subunit of reticulocyte eukaryotic peptide chain initiation factor 2 (eIF-2) from phosphorylation by an eIF-2 kinase, heme-regulated protein synthesis inhibitor (Datta, B., Chakrabarti, D., Roy, A.L., and Gupta, N. K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3324-3328). We now present evidence that this p67 contains multiple O-linked N-acetylglucosamine (GlcNAc) residues, and these glycosyl residues may be required for p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase phosphorylation. Our results are as follows. 1) p67 binds specifically to wheat germ agglutinin, and such binding is completely inhibited in the presence of 0.2 M GlcNAc. 2) The binding of p67 to wheat germ agglutinin leads to complete loss of p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase phosphorylation. 3) p67 accepts 10-12 [3H]galactose molecules from UDP-[3H]galactose in the presence of galactosyltransferase. This radioactivity is resistant to endo-beta-N-acetylglucosamine F (+ peptide:N-glycosidase F) treatment but is completely lost when the 3H-labeled p67 is treated with sodium borohydride in mild alkali (beta-elimination reaction). These results suggest that p67 contains terminal GlcNAc moieties O-linked to the protein. 4) Upon hexosaminidase treatment, p67 reaction product migrated as a lower molecular mass (Mr approximately 65 kDa) protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 5) A monoclonal antibody (D1) against p67 has been isolated. D1 apparently recognizes a specific GlcNAc-containing peptide epitope in p67 and does not react with hexosaminidase-treated p67. These results suggest that p67 activity in the cell may also be regulated post-transcriptionally by glycosylation of p67 protein.
Datta
B
B
Department of Chemistry, School of Biological Sciences, University of Nebraska, Lincoln 68588-0304.
Ray
M K
MK
Chakrabarti
D
D
Wylie
D E
DE
Gupta
N K
NK
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D003126:Colostrum / Q000201:enzymology; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism; D006026:Glycoside Hydrolases; D006031:Glycosylation; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000378:metabolism; D019892:eIF-2 Kinase
D000911:Antibodies, Monoclonal; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D014909:Wheat Germ Agglutinins; D005700:Galactosyltransferases; D011494:Protein Kinases; D019892:eIF-2 Kinase; D006026:Glycoside Hydrolases