N-GLYCOSYLATION DOES NOT AFFECT ASSEMBLY AND TARGETING OF PROGLYCINININ YEAST

Glycinin, a simple protein, and beta-conglycinin, a glycoprotein, are the dominant storage proteins of soybean and are suggested to be deriv ed from a common ancestor. To investigate why glycinin does not requir e glycosylation for its maturation, we attempted N-glycosylation of pr oglycinin A(1a)B(1b) using site-directed mutagenesis and yeast express ion system. An N-glycosylation consensus sequence Asn-X-Ser/Thr was cr eated at positions 103, 183, 196, 284 and 457 in the variable regions being strongly hydrophilic revealed from the alignment of amino acid s equences of various glycinin-type proteins. Among five mutant proglyci nins (Q103N, H183N, G198T, S284N, N459T), Q103N was fully glycosylated , and H183N and N459T were partly (around 20% of the expressed protein s), whereas others were barely or not glycosylated. The glycosylated p roglycinin was susceptible to endo-beta-N-acetylglucosamidase and N-gl ycanase cleavages. N-glycosylation did not cause inconveniences to pro cessing of signal peptide, assembly into trimers and targeting into th e vacuoles. Thermal and trypsin sensitivity analyses of the glycosylat ed proglycinin suggested that N-linked glycan prevents protein-protein interaction but does not stabilize the protein conformation. The reas on why glycinin does not require N-glycosylation for its maturation is discussed. (C) 1998 Elsevier Science B.V.