In Campylobacter jejuni the sugar 2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose, termed N,N ′-diacetylbacillosamine (Bac2,4diNAc), is the first carbohydrate in the glycoprotein N-linked heptasaccharide. Starting with uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) two enzymes of the general protein glycosylation (Pgl) pathway in C. jejuni (PglF and PglE) have been recently shown to modify this sugar nucleotide to form UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-D-glycopyranose (UDP-4-amino-sugar) [Schoenhofen, I. C., et al. (2006) J Biol Chem 281, 723-732]. PglD has been proposed to catalyze the final step in N,N′-diacetylbacillosamine synthesis by N-acetylation of the UDP-4-amino-sugar at the C4 position. We have cloned, overexpressed and purified PglD from the pgl locus of C. jejuni NCTC 11168 and identified it as the acetyltransferase that modifies the UDP-4-amino-sugar to form UDP-N,N′-diacetylbacillosamine, utilizing acetyl coenzyme A as the acetyl group donor. The UDP-N,N′-diacetylbacillosamine product was purified from the reaction by reverse phase C18 HPLC and the structure determined by NMR analysis. Additionally, the full-length PglF was overexpressed and purified in the presence of detergent as a GST-fusion protein allowing for derivation of kinetic parameters. We found that the UDP-4-aminosugar was readily synthesized from UDP-GlcNAc in a coupled reaction using PglF and PglE. We also demonstrate the in vitro biosynthesis of the complete heptasaccharide lipid-linked donor by coupling the action of eight enzymes (PglF, PglE, PglD, PglC, PglA, PglJ, PglH, and PglI) in the Pgl pathway in a single reaction vessel.Campylobacter jejuni is the Gram-negative enteropathogen identified as the primary cause of gastroenteritis in humans and the most frequent infection to precede the peripheral neuropathy Guillain-Barré syndrome (1-3). Infection of humans generally occurs by ingestion of contaminated livestock or water. Although the mechanism of infection is not clearly understood, production of glycolipids and glycoproteins by the pathogen have been found to influence cell motility, host-cell interactions, and competence for DNA uptake (4-6). As resistance to antimicrobial agents rises, the potential for development of novel therapeutics against enzymes that produce glycoconjugates has intensified efforts targeted at the characterization of their biosynthetic pathways. In C. jejuni four major glycan structures have been identified: lipooligosaccharide (LOS), capsule, O-linked glycan, and N-linked glycan *To whom correspondence should be addressed: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. E-mail: imper@mit.edu, (v) 617-253-1838, (f) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (7)(8)(9)(10). The genes coding for enzymes that synthesize these glycan moieties have been found in clusters throughout the C. jejuni genome. The respective biochemical functions of the gene products have been assigned on the b...

Heparin is a highly sulfated glycosaminoglycan that is used as an important clinical anticoagulant. Monitoring and control of the heparin level in a patient's blood during and after surgery is essential, but current clinical methods are limited to indirect and off-line assays. We have developed a silicon field-effect sensor for direct detection of heparin by its intrinsic negative charge. The sensor consists of a simple microfabricated electrolyte-insulatorsilicon structure encapsulated within microfluidic channels. As heparin-specific surface probes the clinical heparin antagonist protamine or the physiological partner antithrombin III were used. The dose-response curves in 10% PBS revealed a detection limit of 0.001 units͞ml, which is orders of magnitude lower than clinically relevant concentrations. We also detected heparin-based drugs such as the low-molecular-weight heparin enoxaparin (Lovenox) and the synthetic pentasaccharide heparin analog fondaparinux (Arixtra), which cannot be monitored by the existing near-patient clinical methods. We demonstrated the specificity of the antithrombin III functionalized sensor for the physiologically active pentasaccharide sequence. As a validation, we showed correlation of our measurements to those from a colorimetric assay for heparin-mediated anti-Xa activity. These results demonstrate that silicon field-effect sensors could be used in the clinic for routine monitoring and maintenance of therapeutic levels of heparin and heparin-based drugs and in the laboratory for quantitation of total amount and specific epitopes of heparin and other glycosaminoglycans.heparin sensors ͉ label-free sensing ͉ medical devices ͉ enoxaparin ͉ fondaparinux

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) have been implicated in clinically relevant processes such as hemostasis, infection, development, and cancer progression, through their interactions with proteins. Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MSn) were combined to identify and quantify 12 HSGAG disaccharides that can be generated by enzymatic depolymerization with heparin lyases. This technique includes free amine-containing disaccharides that had previously been observed in MSn but not quantified. Our methods use diagnostic product ions from MSn spectra of up to three isomeric disaccharides at once, and up to three sequential stages of MSn in tandem, for the quantitative analysis of the relative percentage of each of these isomers. The isomer quantification was validated using mock mixtures and showed acceptable accuracy and precision. These methods may be applied to the quantification of other isomers by MSn. While each of the 12 disaccharides alone had a linear response to an internal standard in the MS1 spectra, the individual response factors did not remain constant when the concentrations of the other 11 disaccharides in the mixtures fluctuated, due to competition for electrospray ionization. The absolute concentration of one fluctuating isomer was determined out of a constant mixture of the other disaccharides. The rapid, accurate, and sensitive quantification of all isomeric disaccharides may contribute to the eventual sequencing of longer saccharides by MSn, enabling the elucidation of the structure-function relationships of HSGAGs.

Sulfated polysaccharides such as heparin and heparan sulfate glycosaminoglycans (HSGAGs) are chemically and structurally heterogeneous biopolymers that that function as key regulators of numerous biological functions. The elucidation of HSGAG fine structure is fundamental to understanding their functional diversity, and this is facilitated by the use of select degrading enzymes of defined substrate specificity. Our previous studies have reported the cloning, characterization, recombinant expression, and structure-function analysis in Escherichia coli of the Flavobacterium heparinum 2-O-sulfatase and 6-O-sulfatase enzymes that cleave O-sulfate groups from specific locations of the HSGAG polymer. Building on these preceding studies, we report here the molecular cloning and recombinant expression in Escherichia coli of an N-sulfamidase, specific for HSGAGs. In addition, we examine the basic enzymology of this enzyme through molecular modeling studies and structure-function analysis of substrate specificity and basic biochemistry. We use the results from these studies to propose a novel mechanism for nitrogen-sulfur bond cleavage by the N-sulfamidase. Taken together, our structural and biochemical studies indicate that N-sulfamidase is a predominantly exolytic enzyme that specifically acts on N-sulfated and 6-O-desulfated glucosamines present as monosaccharides or at the nonreducing end of odd-numbered oligosaccharide substrates. In conjunction with the previously reported specificities for the F. heparinum 2-O-sulfatase, 6-O-sulfatase, and unsaturated glucuronyl hydrolase, we are able to now reconstruct in vitro the defined exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum and apply these enzymes in tandem toward the exo-sequencing of heparin-derived oligosaccharides.The N-sulfate group is characteristic and unique to heparin sulfate glycosaminoglycans (HSGAGs), 4 which are commonly occurring polysaccharides predominant in proteoglycans (1). HSGAGs are linear polymers with variable repeating disaccharide units and diverse chemical heterogeneity due to the variable positions of O-and N-linked sulfates (2, 3). Other factors contributing to the structural diversity of HSGAGs include the presence of N-linked acetates and possible epimerization at the C-5 carboxylates. The structure-function relationship of this diversity plays out in the dynamic regulation by HSGAGs of various signaling pathways (4), including cell death (5, 6), intercellular communication, cell growth and differentiation (7), and adhesion and tissue morphogenesis (8). Microbial pathogenesis has also been shown to depend upon the HSGAGs that are present as structurally defined binding epitopes on cell surfaces and as part of the extracellular matrix (9, 10). GAG degradation follows an obligatory sequence of depolymerization steps involving multiple enzymes acting in tandem to cleave the HSGAG chain. Most of these enzymes act exolytically (11, 12) but may differ in the extent of their processivity, e.g. when compa...