ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 223, No. 1, May, pp. 254-263, 1983 The Biosynthesis of Crustacean Chitin. Isolation and Characterization of Polyprenol-Linked Intermediates from Brine Shrimp Microsomes’ MICHAEL N. HORST’ Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32610 . Received November 30, 1982, and in revised form January 18, 1983 The biosynthesis of crustacean chitin appears to involve the participation of a lipid- linked intermediate. A microsomal preparation from larval stages of the brine shrimp Artemiu sul&u was found to catalyze the glycosylation of exogenous [“Hldolichol phos- phate, yielding a product which was insoluble in chloroform:methanol (2:l) but soluble in chloroform:methanol:water (10:10:3). Artemia microsomes catalyze the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to a lipid acceptor. After ex- traction of labeled lipids with either chloroform:methanol (2:l) or chloro- form:methanol:water (10:10:3), labeled compounds could be purified by ion-exchange chromatography on DEAE-Sephacel. Mild acid hydrolysis of 3H-N-acetylglucosamine labeled material soluble in chloroform:methanol:water (10:10:3) yielded a series of oli- gosaccharides ranging from 2 to about 8 glycosyl units in size. The larger components were shown to be sensitive to chitinase digestion but resistant to treatment with Lu-mannosidase. Such 3H-N-acetylglucosamine containing compounds, prepared by both in vivo and in vitro procedures, appear to be chitin oligosaccharides. Brine shrimp microsomes also catalyze the transfer of mannose from GDP-mannose to a lipid ac- ceptor. Mild acid hydrolysis of mannosyl lipids soluble in chloroform:methanol:water (10:10:3) yielded oligosaccharides which were sensitive to cY-mannosidase digestion and resistant to treatment with endochitinase. The results suggest 3H-N-acetylglu- cosamine-labeled oligosaccharide-lipids are distinct from the mannose-labeled fraction and may participate in the formation of an endogenous primer for chitin synthesis after their transfer to a protein acceptor. Chitin, a linear polymer of N-acetyl-D- glucosamine P-1,4-linked, is a structural polysaccharide found in yeast, fungi, and nearly all invertebrate phyla. In the ar- thropods, chitin is a major structural com- ponent of the cuticle or exoskeleton and is normally covalently attached to protein (1, 2). Although some investigators believe that the protein is attached after comple- tion of the chitin polymer via crosslinking ’ Supported by Grant GM 30952 from the National Institutes of Health. ‘Present address: Division of Basic Medical Sci- ences, School of Medicine, 1550 College St., Mercer University, Macon, Georgia 31207. reactions (3, 4), other explanations are possible. One alternative is that the pro- tein may be involved in the initiation of chitin synthesis and may play a biosyn- thetic role (5). Previous studies have described the properties of a microsomal enzyme from larval stages of the brine shrimp Artemia salina which extends an existing chitin oli- gosaccharide or endogenous primer (6). After incubation of a microsomal prepa- ration in the presence of [3H]UDP-N-ace- tyl-D-glUCOSamiIE, significant radioactiv- ity was detected in a lipid-soluble fraction after extraction of samples with chloro- form:methanol (2:l) followed by chloro- 0003-9861183 $3.00 Copyright Q 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. 254