Wheat-germ agglutinin is synthesized as a glycosylated precursor.
The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [(35)S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochromec reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with Mr 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of Mr 23 000 and the mature monomer of Mr 18000. When [(3)H]mannose was used in labeling studies, only the polypeptide of Mr 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of Mr 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745-6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.
MSU-DOE Plant Research Laboratory, Michigan State University, 48824-1312, East Lansing, MI, USA.
M A
MA
Mansfield
W J
WJ
Peumans
N V
NV
Raikhel