Rapid Chemoenzymatic Synthesis of Monodisperse Hyaluronan Oligosaccharides with Immobilized Enzyme Reactors

Abstract

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...

Synchronized Chemoenzymatic Synthesis of Monodisperse Hyaluronan Polymers

“…A useful property of pmHAS is that it can extend exogenously provided HA acceptor oligosaccharides in vitro (16). This polymerization reaction occurs in a nonprocessive fashion where the enzyme binds, elongates, and releases the growing HA chain in a rapid repetitive fashion (13,17). In this report, we utilize the acceptor elongation activity for the chemoenzymatic synthesis of a variety of HA polymers with narrow size distributions.…”

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

“…Overall, PmHAS appears to be a polypeptide with two active sites involved with coordinated but intrinsically nonprocessive activities. In several indirect tests, including in vitro syntheses of oligosaccharides [21] and polysaccharides [29], PmHAS appears to operate in a nonprocessive fashion. In vivo, PmHAS is probably docked with the membrane transport machinery which may not allow the HA chain to diffuse far away from the microenvironment of the two active sites.…”

“…For MALDI-ToF MS and SEC-MALLS analyses, a preparation of PmHAS 1-703 was purified by chromatography on Toyopearl Red AF resin (Tosoh Corp., Tokyo, Japan) using salt elution (50 mM HEPES, pH 7.2, 1 M ethylene glycol with 0 to 1.5 M NaCl gradient in 1 hour) as described previously [21]. The fractions containing the target protein (~95% pure by SDS-PAGE/Coomassie-staining) were pooled and concentrated by ultrafiltration.…”

An Engineered Hyaluronan Synthase

“…rhHAS2-(122-414) could not synthesize longer (high molecular weight) HA chains even by using longer incubations (over 24 h) or reactions with higher UDP-sugar concentrations (data not shown). Incubation of the tetrasaccharide acceptor with a single donor, UDP-GlcNAc, did not yield HA5, suggesting that the recombinant truncated HAS2 polypeptides cannot be utilized for the stepwise synthesis of HA performed by using the engineered PmHAS method reported by DeAngelis et al (25). For the purpose of the regulation of the molecular size of the HA, some improvement or optimization of the reaction conditions will be required.…”

“…It has also been reported that PmHAS added new sugar residues to the saturated tetramer prepared by means of ovine testis hyaluronidase at the non-reducing end (17). Moreover the recent work of DeAngelis et al (25) clearly demonstrated that the engineered PmHAS could be converted into two single action glycosyltransferases, and reactor enzymes immobilized onto solid supports were utilized in an alternating fashion to make pure HA molecules of a single length in a controlled and stepwise manner. In vertebrates HASs, it is known that HAS of Xenopus DG42 expressed in yeast exhibited a significant activity as HA synthase (26).…”