Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life.

Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.

Insights into the structure and function of membrane-integrated processive glycosyltransferases.

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).

The hyaluronan synthase catalyzes the synthesis and membrane translocation of hyaluronan.

Hyaluronan (HA), an extracellular linear polysaccharide of alternating N-acetyl-glucosamine and glucuronic acid residues, is ubiquitously expressed in vertebrates, where it affects a broad spectrum of physiological processes, including cell adhesion, migration and differentiation. The HA polymer is synthesized on the cytosolic side of the cell membrane by the membrane-embedded hyaluronan synthase (HAS). However, the process by which the extremely hydrophilic HA polymer is translocated across the membrane is unknown to date. The bacterial HAS from Streptococcus equisimilis (Se) shares a similar transmembrane topology and significant sequence identity with human HASs and likely synthesizes HA by the same mechanism. We demonstrate that the Se-HAS is both necessary and sufficient to translocate HA in a reaction that is tightly coupled to HA elongation. The purified Se-HAS is reconstituted into proteoliposomes (PLs) where it synthesizes and translocates HA. In vitro synthesized, high-molecular-weight HA remains tightly associated with the intact PLs in sedimentation experiments. Most importantly, the newly formed HA is protected from enzymatic degradation by hyaluronidase unless the PLs are solubilized with detergent, thereby demonstrating that HA is translocated into the lumen of the vesicle. In addition, we show that HA synthesis and translocation are spatially coupled events, which allow HA synthesis even in the presence of a large excess of HA-degrading enzyme. The coupled synthesis and membrane translocation of a biopolymer represents a novel membrane translocation mechanism and is likely applicable to the synthesis of some of the most abundant biopolymers, including chitin and cellulose.

A highly efficient route to enantiomerically pure l-N-Bz-Pmp(t-Bu)2-OH and incorporation into a peptide-based protein tyrosine phosphatase inhibitor.

Phosphonomethyl phenylalanine (Pmp), a nonhydrolyzable mimic of phosphotyrosine, is an important building block in the development of peptide-based PTP inhibitors. We have designed a novel, efficient synthesis of N-Bz-Pmp(t-Bu)2-OH. A Pmp-containing peptide based on a known biological substrate of the tyrosine phosphatase CD45 (Ac-TEGQ-Pmp-QPQP-NH2) inhibits CD45 with an IC50 value of approximately 100 microM with virtually no inhibition of TCPTP up to concentrations of 120 microM.