Swainsonine-induced oligosaccharide excretion in sheep. Time-dependent changes in the oligosaccharide profile

Abstract

Urinary oligosaccharides isolated from locoweed-intoxicated sheep were separated and quantified by reversed-phase high pressure liquid chromatography of the perbenzoylated alditols. Mannose-containing oligosaccharides were elevated as early as day 3 of feeding, but maximum levels (approx. 1 μmol/ml) were not attained until after 6 weeks of feeding. The relative abundance of individual oligosaccharides changed over the course of the feeding period. Man3GlcNAc2 reached a peak on day 3 and then rapidly declined. Two isomers were shown to be present in this fraction and the relative proportions altered with the duration of locoweed treatment. The major isomer present at early time points (<8 days) co-eluted with synthetic Man(α1-3)[Man(α1-6)]Man(β1-4)GlcNAc(β1-4)GlcNac, was digested by endo-β-N-acetylglucosaminidase D, and is probably derived from the trimannosyl core of complex glycoproteins synthesized prior to locoweed treatment. Man3GlcNAc2 isolated from day 53 urine was resistant to endo-β-N-acetylglucosaminidase D digestion but was cleaved by endo-β-N-acetylglucosaminidase H. This isomer has the probable structure Man(α1-3)Man(α1-6)Man(β1-4)GlcNAc(β1-4)GlcNAc, indicative of its origin from hybrid or high-mannose glycoproteins. Man5GlcNAc2 reached a peak on day 13 and then slowly declined, whereas Man4GlcNAc2 increased concomitantly. The rapid increase in Man5GlcNAc2 can probably be attributed to the breakdown of hybrid glycans produced as a result of swainsonine inhibition of Golgi α-D-mannosidase II. The onset of observable clinical signs on day 38 closely correlated with the time point at which the level of Man4GlcNAc2 exceeded Man5GlcNAc2. After locoweed feeding was discontinued, the amount of urinary olgigosaccharides declined rapidly and reached baseline levels within 12 days.