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We determined the complete nucleotide sequence of EDRD-1, a Japanese strain of the North American type-Porcine reproductive and respiratory syndrome virus (PRRSV), and identified a novel 117-base deletion and 108-base insertion previously reported in the nsp2 gene of the SP strain, which contains the largest genome among PRRSV strains. Based on genetic analysis of the partial nsp2 gene in 30 additional Japanese isolates and 50 strains from various countries, we classified North American-type PRRSVs into three nsp2-types, represented by EDRD-1, which contains the 117-base deletion and 108-base insertion; prototypic VR-2332, which does not contain the deletion and insertion; and SP, which contains only the 108-base insertion. The three nsp2-types were phylogenetically separated, suggesting that these structural changes only occurred at earlier stages of viral evolution. In the nsp2 genes, we identified an additional 19 deletions ranging from 3 to 378 bases and 2 insertions of 3 and 21 bases which were not common within each nsp2-type, suggesting that these changes occurred at later stages of viral evolution. In addition, our results suggest that the three nsp2-types can be rapidly differentiated by RT-PCR using their polymorphisms as natural tags.

Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of alpha- and beta-subunits with an identical amino acid sequence but show different biological activities. To elucidate the molecular difference between these gonadotropins, the structure of the N-linked oligosaccharides of each beta-subunit was determined. N-linked sugar chains, liberated as tritum-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and reduction with NaB3H4, were neutralized by sialidase digestion and/or methanolytic desulfation. Neutralized oligosaccharides were fractionated by sequential chromatography on serial lectin affinity columns and on a Bio-Gel P-4 column. Each oligosaccharide structure was determined by sequential exoglycosidase digestion in conjunction with elution profiles on lectin columns and methylation analysis. Each beta-subunit contained a single N-glycosylation site, but a high degree of microheterogeneity was observed in the structure of its N-linked oligosaccharides. eCG beta contained mono-, bi-, tri-, and tetraantennary complex-type oligosaccharides in a ratio of 3:63:13:1. eCG beta oligosaccharides contained about 16% of the bisecting GlcNAc and about 20% of poly-N-acetyllactosamine structures. Elongation of N-acetyllactosamine units showed a preference to the Man alpha 1-->6 side rather than the Man alpha 1-->3 side. Triantennary chains had only a C-2, 4-branched structure. eLH beta contained only mono- and biantennary complex-type and hybrid-type oligosaccharides in a ratio of approximately 18:67:10. eLH beta also contained bisected structures in about 18%. Oligosaccharides derived from the sulfated fraction of eLH beta contained GalNAc residues at nonreducing termini. Oligosaccharides from the sialylated/sulfated fraction of eLH beta contained both Gal and GalNAc residues at nonreducing termini, and those GalNAc residues were preferentially distributed to the Man alpha 1-->3 side of the trimannosyl core. These results clearly indicate that eCG beta and eLH beta possess structurally distinct N-linked oligosaccharides in addition to different charge groups even though they have a protein moiety identical to each other. Our results suggest that the biological activity of these hormones might be modulated by its terminal charge groups and stem structures of carbohydrate moiety synthesized in different organs.

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The thermostable alpha-galactosidase from Candida guilliermondii H-404 synthesized self-transfer products in the absence of a suitable acceptor. The main self-transfer product, using melibiose as a donor substrate, was O-alpha-D-galactosyl-(1,6)-O-alpha-D-galactosyl-(1,6)-D-glucose. This enzyme had a wide acceptor specificity. D-Glucose, D-galactose, maltose, maltitol, and 1,4-butandiol were the most effective acceptors in the transgalactosylation catalyzed by this enzyme. The enzyme could also transfer alpha-galactosyl residues to pentoses (L-arabinose, D-xylose, and D-ribose) and methyl pentoses (D-fucose and L-rhamnose). The main transfer products to lactose, maltose, and sucrose as acceptors were identified as O-alpha-D-galactosyl-(1,6)-O-beta-D-galactosyl-(1,4)-D-glucose, O-alpha-D-galactosyl-(1,6)-O-alpha-D-glucosyl-(1,4)-D-glucose, and O-alpha-D-galactosyl-(1,6)-O-alpha-D-glucosyl-(1,2)-beta-D-fructoside (raffinose), respectively.