science/technology
GLYCOPROTEIN SYNTHESIZED Researchers use enzymatic approach to make 'glycoform' of ribonuclease Β containing branched oligosaccharide StuBorman C&EN Washington
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sing two different strategies, bio chemists at Scripps Research Insti tute, La Jolla, Calif., have enzymatically synthesized a glycoprotein with a modified oligosaccharide group. Graduate student Krista Witte, postdoc toral associates Pamela Sears and Richard Martin, and chemistry professor Chi-Huey Wong used an engineered version of the enzyme subtilisin and various glycosyltransferase enzymes to synthesize glycoforms of the protein ribonuclease Β [/. Am. Chem. Soc, 119, 2114 (1997)]. The work shows once again that syn thetic ingenuity can be used to create natural and nonnatural biomolecules of virtually any size and complexity in the laboratory. Glycopeptides and glycoproteins (peptides and proteins with attached car bohydrate chains) are among the most complex natural compounds. They play a vital role in virology, immunology, bio logical recognition, biomolecular trans port, and other biological processes. Glycopeptides have been synthesized before. But the Scripps study is the first in which a glycoprotein has been assem bled from fragments and a new carbohy drate group has been added. According to Samuel J. Danishefsky, director of the Laboratory for Bioorganic Chemistry at Sloan-Kettering Institute for Cancer Research, New York City, and professor of chemistry at Columbia Uni versity, who specializes in carbohydrate chemistry, the report "is one of the most interesting and exciting papers in the carbohydrate field which I've had the oc casion to read in some time. In this work, Wong has brought to full fruition his brilliant orchestrations of enzymatically mediated syntheses of oligosaccha ride and oligoconjugate domains. That he was able to do this in the context of creating new glycosidic variations of ri ΊΔ.
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remove the heterogeneous sugar moieties, leaving one sugar (7V-acetylglucosamine) as an attachment point. Glycosyltransferase enzymes are then used to add selected sugars to the protein's glycosylation site. The researchers synthesized a new ribonu clease Β glycoform containing the branched oligosaccharide sialyl Lewis X to demon strate this route. The second strategy is a combined chemical and enzymatic technique. Pep tides and a glycopeptide are first synthe sized by solid-phase chemical synthesis. The pieces are joined enzymatically us ing engineered subtilisin, and the oligo saccharide originally present on the gly-
bonuclease Β is a most impressive dem onstration of the power and versatility of his methodology." Glycoproteins (such as the anemia drug erythropoi etin) can be produced by re combinant DNA techniques, but such in vivo methods produce complex mixtures of many different versions (glycoforms) that differ in carbohydrate content. Indi vidual glycoforms can be sep arated out of those mixtures by analytical separation tech niques, but such processes "are very complicated and tedious and are limited to use with natural glycopro teins," according to Wong and coworkers. Wongand coworkers added the tetrasaccharlde sialyl In the JACS paper, Wong Lewis X (above, bottomright)to the glycosylation site of and coworkers present two ribonuclease Β to create this modified glycoprotein. strategies for making pure ri Protein chain ofribonucleaseis white and yellow, disulfide bonds and dlsulflde-linked residues are pink, and bonuclease Β glycoforms. The glycosylation site (an asparagine residue) Is green. In first is to take glycoproteins sialyl Lewis X, carbon is green, oxygen is red, and produced recombinantly and nitrogen is blue.
Two synthetic routes to a glycoprotein
The glycoprotein synthesis follows a string of related achievements in glycopeptide synthesis over the past few years. Organic chemist Horst Kunz of Johannes Gutenberg University, Mainz, ral ones that can be Germany, conceived key protectingmade by the first group strategies that have been used to synthesize glycopeptides. Danishefsky, method. Wong believes Wong, chemistry professor Daniel E. that the ability to Kahne of Princeton University, and seobtain pure glyco- nior scientist Morten Meldal of Carlsberg proteins of defined Laboratory, Valby, Denmark, have develcomposition more oped techniques for solid-phase syntheeasily, via enzymatic sis of glycopeptides. Other key contributors to the field of synthesis, could lead to the development glycosynthesis include Bertram O. Fraserof better glycopro- Reid, president and director of Natural tein-based drugs and Products & Glycotechnology Research facilitate basic re- Institute, Research Triangle Park, N.C., search on the struc- and chemistry professors Hans Paulsen tural and functional of the University of Hamburg (Germany), role of carbohydrates and Klaus Bock of Carlsberg Laboratory. in proteins. "People are interested in gly- In addition, biochemist James A. Wells of coprotein folding, structure, and activi- the department of protein engineering at ty, and the effects of sugars on protein Genentech Inc., South San Francisco, has structure and function," he says. "You achieved the total synthesis of a nonglyneed to have a tool to address these cosylated ribonuclease A using another engineered subtilising problems." Wong: interest in glycoprotein folding, structure, and activity
eptide is used as an attachment site the glycosyltransferase-catalyzed addiof new sugars. Wong says the second egy is the more important of the two mse it can be used to produce nonnatglycoproteins in addition to the natu-
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