Volume 139, number 2 FEBS LETTERS March 1982 zyxwvutsr CHARACTERIZATION OF A NEW ISOMER OF LIPID-LINKED HEPTASACCHARIDE FORMED DURING IN VITRO BIOSYNTHESIS OF MAMMARY GLYCOPROTEINS Inder K. VIJAY and Gary H. PERDEW Department of Dairy Science, University of Maryland, College Park, MD 20742, USA Received 1 February 1982 1. Introduction Incubation of microsomes from the lactating bovine mammary tissue with UDP-GlcNAc and GDP-Man results in the biosynthesis of lipid-linked oligosaccha- rides Man,r(GlcNA~)~, n = l-9 [I]. Pulse and chase kinetics indicated these to be interrelated as precur- sor-products for the biosynthesis of asparagine- linked glycoproteins in this tissue. Structural analyses showed that among these, Man(GlcNAch through ManS(GlcNAc)z and Man,(GlcNAc)* species were monoisomeric [ 11;however, 2 isomers of Maq(GlcNAc), and 3 isomers of Mans(GlcNA~)~ could be iden tidied [2]. The resolution of isomers among hexa- and hepta- saccharides was facilitated by the specificity of endo D and endo H towards oligomannosylchitobiose sub- strates [3,4]. The heptasaccharide cleaved by endo D was characterized as Manal+2Manal+3Maml+6- (Mancul+3)Mar@l+4(3)GlcNAcf31+4(3)GlcNAc. Structural data on the octa- through decasaccharide indicated that an additional isomer might also be present in the endo-Dcleaved heptasaccharide. Using controlled acetolysis, in which only incipient cleavage of the a-1,6 linkages occurs and aided by the availabil- ity of an cr-1,2-specific mannosidase, we now report the characterization of another isomer within the heptasaccharide, Mans(GlcNAc),. Abbreviations: Man, mannose; GlcNAc, N-acetylglucosamine; (GlcNAc),, NJ/‘-diacetylchitobiose; subscript OH and OT refer to NaBH,- and NaBsH,-reduced oligosaccharides; endo, endop-N-acetylglucosaminidase; CHO, Chinese hamster ovary; AlI sugars are of the Dconfiguration 2. Materials and methods These were as in [ 11, with the exception of minor additions and changes noted below. The [ 14C]mannose-labeled heptasaccharide isolated from a large scale in vitro incubation run without exogenous dolichol phosphate [2] was digested with endo D as before. The cleaved fragment MansGlcNAc was separated from the resistant ManS(GlcNAc), by gel filtration on column of Bio Gel P-4 [ zyxwvutsrqponmlkj 1] and is the source material for these results. 2.1. Digestion with ac-I ,2-mannosidase This enzyme was isolated from Aspergillus saitoi [5,6]. it had no o-l,3 and c&l,6 mannosidase activ- ity since it failed to digest [ 14C] mannose-labeled Mancul~3(Mancu1+6)Man~l+4(3)GlcNAq?l+4(3)- GlcNAc. [ r4C]Mannose-labeled Ma+,GlcNAc (-30 000 cpm) was treated with 100 ng enzyme in 0.05 M sodium acetate, pH 5.0 at 37’C under toluene. Additional enzyme was added after 24 and 48 h and the incubation continued for 72 h. After desalting [ 11, the products were chromatographed on Schleicher and Schiill paper 589C in ethyl acetatelpyridinelacetic acid/water (5: 5 : 1:3 by vol.) for 33 h. 2.2, Acetolysis _ This was conducted as before except that the incu- bation temperature was 30°C and the reaction was run for either 3 h (incipient acetolysis) or for 7 h (extended acetolysis). Incipient acetolysis revealed no cleavage of o-1,2, o-l,3 and /I-1,4 linkages in Mancw1+2Mancrl+2Mana1+3Mar@1+4GlcNAcoT [I] and [ r4C]mannose-labeled M~QG~cNAcoB character- ized earlier [ 11. Only a trace release of free mannose Atblished by Elsevier Biomedical Press 00145793/82/0000-0000/$02.75 0 1982 Federation of European Biochemical Societies 321