Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan having a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike the template driven synthesis of nucleic acids and proteins, Golgi synthesized glycosaminoglycans are not believed to have predictable or deterministic sequence. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size similar and charge similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular-ions corresponding to mono-compositional peptidoglycosaminoglycans. Fragmentation using collision induced dissociation surprisingly afforded a single sequence for each mono-compositional parent-ion, unequivocally demonstrating the presence of a defined sequence. The common biosynthetic pathway for all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment of our understanding of information storage in complex glycans.
Heparosan is an acidic polysaccharide natural product, which serves as the critical precursor in heparin biosynthesis and in the chemoenzymatic synthesis of bioengineered heparin. Heparosan is also the capsular polysaccharide of Escherichia coli K5 strain. The current study was focused on the examination of the fermentation of E. coli K5 with the goal of producing heparosan in high yield and volumetric productivity. The structure and molecular weight properties of this bacterial heparosan were determined using polyacrylamide gel electrophoresis and Fourier transform-mass spectrometry. Fermentation of E. coli K5 in a defined medium using exponential fed batch glucose addition with oxygen enrichment afforded heparosan at 15 g/L having a number average molecular weight of 58,000 Da and a weight average molecular weight of 84,000 Da.
Seven pharmaceutical heparins were investigated by oligosaccharide mapping by digestion with heparin lyase 1, 2 or 3, followed by high performance liquid chromatography analysis. The structure of one of the prepared mapping standards, ΔUA-Gal-Gal-Xyl-O-CH 2 CONHCH 2 COOH, (where ΔUA is 4-deoxy-α-L-threo-hex-4-eno-pyranosyluronic acid, Gal is β-D-galactpyranose, and Xyl is β-D-xylopyranose) released from the linkage region using either heparin lyase 2 or heparin lyase 3 digestion, is reported for the first time. A size-dependent susceptibility of site cleaved by heparin lyase 3 was also observed. Heparin lyase 3 acts on the under sulfated domains of the heparin chain and does not cleave the linkages within heparin's antithrombin III binding site. Thus, a novel low molecular weight heparin (LMWH) was afforded on heparin lyase 3 digestion of heparin due to this unique substrate specificity, which has anticoagulant activity comparable to that of currently available LMWH.
One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and se-
Glycosaminoglycans (GAGs) are a class of biologically important molecules and their structural analysis is the target of considerable research effort. Advances in tandem mass spectrometry (MS/MS) have recently enabled the structural characterization of several classes of GAGs. However, the highly sulfated GAGs, such as heparins, have remained a relatively intractable class due their tendency to lose SO3 during MS/MS producing few sequence-informative fragment ions. The present work demonstrates for the first time the complete structural characterization of the highly sulfated heparin-based drug, Arixtra. This was achieved by Na+/H+ exchange, to create a more ionized species that was stable against SO3 loss, and that produced complete sets of both glycosidic and cross-ring fragment ions. MS/MS, enabling the complete structural determination of Arixtra®, including the stereochemistry of its uronic acid residues and suggests an approach for solving the structure of more complex, highly sulfated heparin-based drugs.
Proteoglycomics is a systematic study of structure, expression, and function of proteoglycans, a posttranslationally modified subset of a proteome. Although relying on the established technologies of proteomics and glycomics, proteoglycomics research requires unique approaches for elucidating structure-function relationships of both proteoglycan components, glycosaminoglycan chain, and core protein. This review discusses our current understanding of structure and function of proteoglycans, major players in the development, normal physiology, and disease. A brief outline of the proteoglycomic sample preparation and analysis is provided along with examples of several recent proteoglycomic studies. Unique challenges in the characterization of glycosaminoglycan component of proteoglycans are discussed, with emphasis on the many analytical tools used and the types of information they provide.
Proteoglycans (PGs) are among the most structurally complex biomacromolecules in nature. They are present in all animal cells and frequently exert their critical biological functions through interactions with protein ligands and receptors. PGs are comprised of a core protein to which one or multiple, heterogeneous, and polydisperse glycosaminoglycan (GAG) chains are attached. Proteins, including the protein core of PGs, are now routinely sequenced either directly using proteomics or indirectly using molecular biology through their encoding DNA. The sequencing of the GAG component of PGs poses a considerably more difficult challenge because of the relatively underdeveloped state of glycomics and because the control of their biosynthesis in the endoplasmic reticulum and the Golgi is poorly understood and not believed to be template driven. Recently, the GAG chain of the simplest PG has been suggested to have a defined sequence based on its top-down Fourier transform mass spectral sequencing. This review examines the advances made over the past decade in the sequencing of GAG chains and the challenges the field face in sequencing complex PGs having critical biological functions in developmental biology and pathogenesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.