Due to differential sulfation, domain structure (i.e., nonuniform distribution of disaccharides), and variable polysaccharide length, the structure and sequence of heparan sulfate (HS) is highly variable. This structural variability arises mostly from the fact that HS is synthesized in a nontemplate manner, through the concerted action of biosynthetic enzymes, including the O-sulfotransferases, the N-deactylase/N-sulfotransferase, and the C5-epimerase (Salmivirta et al. 1996
). However, this variability, while significant, is controlled, through the tissue/cell-specific expression of enzyme isoforms. This has been confirmed experimentally by examining HS composition across different tissue preparations (Guimond et al. 2009
; Shi and Zaia 2009
) where there are primarily six major disaccharides (), with the most abundant being a nonsulfated uronic acid linked to an N-acetylglucosamine (I/G → HNAc
). In this context, heparin can be thought of as a “specialized” HS, one that is more highly sulfated, and hence contains less variability. Especially in the context of HS, the challenge of detailed structural analysis is exacerbated by the fact that, because synthesis is nontemplate-driven, strategies employed for other template-driven biopolymers, such as DNA or proteins, are not amenable to sequencing complex polysaccharides. Thus, new glycan-specific techniques must either be developed de novo or existing techniques (e.g., proteomic-based techniques) must be substantially adapted.
Six disaccharides that constitute the major building blocks of heparan sulfate chains
Numerous efforts have been made to facilitate the sequencing of heparin and HS chains, primarily to determine structure/function relationships for important protein-binding saccharides. In recent years, these efforts have been enhanced through the development of sophisticated analytical tools, including mass spectrometry and nuclear magnetic resonance (Kuberan et al. 2002
; Yates et al. 1996
; Saad et al. 2005
). Additionally, heparin-degrading enzymes, primarily the bacterially derived heparinases, but also the mammalian enzyme heparanase, have proven to be important tools for sequencing of heparin and HS (Bisio et al. 2007
Broadly, efforts to provide structural information for heparin and HS include both top-down approaches, where key structural information is obtained on intact chains, and bottom-up approaches, where either partial or complete digestion of the chains is carried out, followed by identification /quantification of the resulting fragments. In terms of top-down approaches, monodimensional (1
H and 13
C) and multidimensional NMR have been employed. Proton and 13
C NMR have been used to differentiate animal source and key structural motifs of heparin and HS (Guerrini et al. 2001
). Additionally, for shorter heparin fragments, up to tetradecasaccharides, multidimensional NMR can be used to obtain complete sequence information. In the case of complex mixtures, including intact heparin and low-molecular-weight heparin, multidimensional NMR can be used to identify mono- and disaccharide constituents as well as unusual building blocks produced in low-molecular-weight heparins as a function of the process () (Guerrini et al. 2007
). Notably, multidimensional NMR was the key experimental technique to identify the structure of the heparin contaminant, oversulfated chondroitin sulfate (Guerrini et al. 2008
Fig. 4 HSQC spectra of low-molecular-weight heparins. 2D-NMR spectra of dalteparin and enoxaparin exhibit unique cross peaks arising from signature structures. These structures result from the specific chemistry used to depolymerize unfractionated heparin into (more ...)
In addition to recent advances in NMR analysis of heparin/HS, mass spectrometry has been used to examine the fine structure of heparin and HS. While information from intact analysis of heparin chains is limited, in-line placement of high-resolution gel permeation chromatography has been successfully used to determine composition of low-molecular-weight heparin chains up to octadecasaccharide (Henriksen et al. 2004
). Additionally, such an approach has recently been used to determine detailed structural information for the chondroitin sulfate proteoglycan, bikunin (Chi et al. 2008
Coupled with the above advances, progress has also been made in strategies to fragment HS chains. Historically, application of high energy sufficient for fragmentation (and even ionization) has typically resulted in desulfation and the production of low information content fragments. These limitations have been overcome through several strategies, including formation of noncovalent complexes with cationic buffers or basic peptides (Juhasz and Biemann 1994
; Rhomberg et al. 1998
), application of gentle ionization conditions, as well as alternative strategies for fragmentation, including electron detachment dissociation (EDD) (Wolff et al. 2007
). These advances have led to strategies where informative ions, including B and Y ions () can be formed and detected. Such approaches have been used to sequence heparin-derived saccharides. However, there are limitations with these approaches; while sequence information has been obtained for sequences up to hexasaccharides, sequencing longer fragments still suffers from loss of structural information due to substantial sulfate loss during fragmentation.
Fig. 5 Sequencing of heparin oligosaccharides by MS fragmentation. Several researchers have focused on developing MS-based sequencing procedures for heparin oligosaccharides. Shown here is different potential fragments for a representative tetrasaccharide: I (more ...)
Bottom-up approaches, involving either partial or complete degradation of the heparin/HS chains and determination of the monosaccharide constituents of the fragments has been largely perfected. Quantification is typically completed through separating the resulting fragments by ion exchange or ion-pairing reverse phase HPLC or via capillary electrophoresis. Identification is typically completed either through co-injection with reference standards or through MS and MSn
analysis by online coupling, especially for ion-pairing HPLC where labile pairing agents, such as dibutylamine, have simplified detection (Kuberan et al. 2002
). An alternative strategy that has been employed is direct infusion of a digested mixture and detection and quantification by MS (Saad and Leary 2003
In terms of digestion strategies, chemical means such as nitrous acid have been successfully used to determine disaccharide composition and have the added advantage that such strategies maintain the epimeric state of the uronic acid. However, the fact that no chromophore is generated and that detection typically must be completed through labeling (most often using radioactive sodium borohydride) has limited the adoption of this technique. An alternative strategy, employing bacterially derived heparinases, has been more widely adopted. Here, addition of one or more heparin-degrading enzymes, each with distinct, but overlapping substrate specificity, results in cleavage of heparin or HS (Ernst et al. 1995
). Both complete digestion down to di- and tetrasaccharides, and partial digestion have been completed (Linhardt et al. 1988
; Karamanos et al. 1997
). Since these enzymes are lyases, digestion results in the formation of a Δ4,5 double bond on the uronic acid. Such species absorb UV light strongly, providing a tag for detection. Furthermore, structural information, specifically the presence or absence of an N-acetyl moiety, can be determined. Also of benefit is that the digestion can be completed at room temperature at pH of around 7, conditions conducive with the retention of labile sulfates. Alternatively, detection can be achieved through labeling the reducing end via reductive amination. Such approaches have dramatically increased sensitivity, especially in conjunction with laser-induced fluorescence, enabling detection of femtomoles of material.
Finally, given their high charge density and isomeric configurations (as well as the presence of α and β isoforms), often saccharide components will co-elute/co-migrate with one another. This greatly complicates both identification and quantification. While MS can be employed online to differentiate isomeric states, an alternative, easily implemented strategy is the use of additional bacterial or mammalian degradation enzymes, including the Δ4,5 glycuronidase (Myette et al. 2002
), and the 2-O and 6-O sulfatases (Myette et al. 2003
). Concerted or stepwise addition of these enzymes has been used to resolve di- and oligosaccharides, enabling accurate quantification and determination of mass balance.
Given the structural complexity of heparin and HS, as well as the increasingly sophisticated insight that we are obtaining into biological processes, it is likely that integration of several independent approaches, including both top-down and bottom-up approaches will prove important. Thus, while some methodologies, such as those outlined above, have provided superior information content, dataset integration enables more complete and rapid assessment of sequence and structure–function than any individual method used in isolation. As such development of frameworks to enable integration of datasets from multiple techniques will continue to prove important for the sequencing of heparin and HS (Venkataraman et al. 1999
). As one example of such an integrative approach, results from NMR-based analysis of heparin fragments has been complemented and extended by CE-based disaccharide analysis after digestion with a cocktail of enzymes. In this manner, use of both datasets in an iterative and integrative manner enables one to “walk” through the saccharide sequence (Guerrini et al. 2002
While the above approach has been successfully applied to isolated saccharides, or simple mixtures, for more complex mixtures, such as what has been isolated from cell surface HS, use of multiple approaches, or constraints, will likely be necessary; however, the approach is still valid. Depending on the complexity of the mixture, use of multiple, orthogonal constraints will likely be necessary, including both top-down and bottom-up approaches. Indeed, recently such logic was employed by the U.S. FDA in the approval of the first generic low-molecular-weight heparin.