We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (␤4-glucuronic acid-␤3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (ϳ10 -20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (ϳ1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicanttagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.Complex carbohydrates play many essential roles in vertebrates, but these molecules are often difficult to isolate or to synthesize in pure form and in large amounts for study. HA, 1 a member of the glycosaminoglycan polysaccharide family that includes heparin and chondroitin, is a repeating ␤4-glucuronic acid-␤3-N-acetylglucosamine polymer that is prevalent in the vertebrate body. HA has numerous essential roles in mammals, including modulation of cellular adhesion, signaling, and motility (1, 2). The polymer is thought to be synthesized initially as a chain of ϳ10 5-6 Da (ϳ10 3-4 sugars), which then degrades during turnover in the body to generate a variety of smaller polymers. Although the mechanisms are not completely understood, it has been observed that the size of the HA polymer dictates its effect on cells. Various cell-surface receptors or binding proteins, termed hyaladherins, are postulated to sense the presence and the size of HA (3-5). Hyaladherins thought to play important roles include CD44 and RHAMM. Multivalent binding and/or multimerization of receptors may potentially occur with large polymers but not with small chains, resulting in the alteration of various signaling events.Recently, several groups have reported that HA oligosaccharides (ϳ2-3.5 kDa or ϳ10 -17 sugars) have extremely interesting effects on cellular behavior. These small sugar molecules induce a variety of cancer cells to undergo programmed cell death or apoptosis (6, 7). This intriguing finding immediately suggests the use of HA oligosaccharides as an aid to chemotherapy or radiation treatments to reduce tumor load or limit...

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal␣1,Gal␣1,3Fuc␣1,2Gal-␤1,3GlcNAc␣1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal␣1,3Fuc linkage by transfer of Gal from UDP-␣Gal to Fuc␣1,2Gal␤1,3GlcNAc␣1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal ␤-propeller domain. Overexpression of GT78 with a His 6 -tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His 6 -GT78 exhibited ␣3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 ␣3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.