Characteristics of oligosaccharides from rat parotid (RP) N-linked glycoproteins (GP) after. beta. -adrenoreceptor (. beta. -AR) stimulation

The authors have shown that ..beta..-AR stimulation of RP cells leads to marked enhancement of N-linked glycosylation in 4 secretory GP (Mrapprox.220Kd, HMW;approx.32-38Kd, MMW;approx.17Kd, LMW). To characterize oligosaccharides in GP, cells were incubated 60 min +/- isoproterenol (ISO) and analyzed 2 ways. First, cell extracts were subjected to SDS-PAGE and Western blotting with peroxidase-conjugated Con A or wheat germ agglutinin (WGA). Second, double-labeled (/sup 3/H) man//sup 14/C leu) extracts were chromatographed on G200 followed by analysis of GP on Con A-Sepharose. HMW from control (CON) and ISO cells were Endo H insensitive, Endo F sensitive, altered by incubation with deoxynojirimycin (dNM), and bound both Con A and WGA conjugates. Similar findings were observed with LMW while MMW were sensitive to Endo H and Endo F, unaffected by dNM, bound Con A (strongly) and WGA (weakly). MMW and LMW were primarily eluted from Con A-Sepharose with 0.5M ..cap alpha..-methyl mannoside (..cap alpha..-MM) while HMW were eluted sequentially with 10 mM ..cap alpha..-methyl glucoside and ..cap alpha..-MM. HMW, MMW, and LMW had approx.4 fold higher /sup 3/H//sup 14/C ratios after ISO. These results suggest HMW and LMW likely contain biantennary complex and hybrid oligosaccharides while MMW contain only high mannose oligosaccharide types.