MinireviewNMR structural determination of unique invertebrate glycosaminoglycans endowed with medical properties
Graphical abstract
Introduction
Glycosaminoglycans (GAGs) are glycans of diverse functions and complex structures, although composed of disaccharide building blocks. These repeating blocks are in turn composed of alternating uronic acid and hexosamine monosaccharide units, which can be differently sulfated depending on the GAG families. Members of the same GAG family share common structural features. For example, heparan sulfate (HS) and heparin (Hp) are composed of α-d-glucosamine (GlcN) units as hexosamine type. While HS is less modified by N- and O-sulfation and uronic acid C5 epimerization, which converts the composing β-d-glucuronic acid (GlcA) into α-l-iduronic acid (IdoA); Hp is highly processed by these modifications.1, 2 The other two biologically active GAGs highly abundant in cell surface or extracellular matrix-associated proteoglycans are the chondroitin sulfate (CS) and dermatan sulfate (DS). Although CS and DS are both composed of N-acetyl-β-d-galactosamine (GalNAc) units, the former is made up primarily of GlcA whereas IdoA composes the latter.3
Due to this great structural variety and presence of sulfation, GAGs can bind to an incredible number of functional proteins, including, but not limited to, inflammatory cytokines and chemokines,4 angiogenesis-related growth factors like fibroblast growth factors 1,5 and 2 (FGF2),6 and plasma cofactors involved in blood coagulation like thrombin (IIa),7 and antithrombin (AT).7, 8 Because of these and other unmentioned roles, GAGs are ubiquitously distributed in all vertebrates usually linked to a protein core forming thus the well-known proteoglycans. Structural and functional analyses of GAGs from mammals are largely carried out worldwide given to the fact that these molecules play key roles in multiple pathophysiological processes such as cancer,9, 10 inflammation,11 thrombosis,12 and neovascularization.13 The mechanisms of action of GAGs in these events are directly related to their structural features.
However, GAGs of medical utilities are not derived exclusively from mammals. Therapeutic GAGs from invertebrates, from terrestrial or marine habitat, are also available and have shown curiously different structures as compared to the mammalian standards. Among numerous invertebrate GAGs that have been studied so far in terms of both structure and function, representative examples are the holothurian fucosylated chondroitin sulfate (FucCS),14, 15 the acharan sulfate (AS) from the giant African snail Achatina fulica,16, 17, 18 the ascidian DSs of unique sulfation patterns,19, 20, 21 the novel HS from the bivalve Nodipecten nodosus,22 and the hybrid HS/Hp found in the head of the shrimp Litopenaeus vannamei.23 Like in structural analyses of mammalian GAGs, solution nuclear magnetic resonance (NMR) spectroscopy alone or combined with other techniques, has been the preferred method used in structural characterization of the invertebrate molecules. Here, this NMR-based analysis of GAGs of unique structures found exclusively in invertebrate animals is being overviewed in order to offer a straightforward technical guidance to future researches in the area. Discussion about the medicinal implications of these distinct invertebrate GAG structures is also provided in order to highlight their potential benefits in future treatments and prophylaxis of certain diseases.
Section snippets
Holothurian FucCS
The first evidence describing the existence that sea cucumbers (Echinodermata, Holothuroidea) could synthesize a different GAG in their body wall seems to come from reference 24. Through monosaccharide composition analyses, the new GAG extracted from the holothurian species Ludwigothurea grisea was reported to be composed of equimolar quantities of α-l-fucopyranose (Fucp), β-d-GlcA and β-d-GalNAc units.25 In a further work of the same group, the investigators have expanded the structural
Terrestrial mollusk AS
The first description of a novel GAG in the giant African snail (terrestrial pulmonate gastropod mollusk) A. fulica seems to be reference 16. As implied, the name acharan sulfate (AS) derives from the scientific name of the invertebrate species who synthesizes this GAG. Based on NMR analyses, the AS structure was shown to be very homogeneous in terms of both chemical substitution and monosaccharide variation. These features are somewhat atypical for GAG-like molecules. Nonetheless, in light of
Ascidian DS
Although DS is composed of repeating disaccharide building blocks made up of alternating 4-linked α-l-IdoA and 3-linked β-d-GalNAc units, sulfation patterns can vary among DS from different tissues, cells or pathophysiological conditions.3 Nonetheless, in the commonest DSs from mammals, while GalNAc are mostly sulfated at the C4 position (∼95%) and slightly at the C6 position (∼15%); composing IdoA units is just occasionally sulfated at the C2 position (∼5%) (Fig. 5). In ascidians, also known
Marine mollusk HS
HS is composed of alternating α-d-GlcN and an uronic acid (either β-d-GlcA, mostly, or α-l-IdoA, occasionally). All monosaccharides present glycosidic bonds at the C4 positions. The amino sugars can be further modified by O-sulfation at C6 (frequently) and C3 (very rarely) or by N-sulfation at the amino group (frequently).2 Besides the intrinsic epimerization process of the uronic acid units, which leads to the clear distinction of β-d-GlcA and α-l-IdoA, these units can be further processed by
Crustacean Hs/Hp
As briefly introduced above, Hp and HS share basically the same structural disaccharide precursor [→4)-β-d-GlcA-(1→4)-α-d-GlcNAc-(1→] (Fig. 9A). However, after the polymerization process during their biosyntheses, different degrees of chain modification occur to each GAG type.1, 2 HS is less processed by the biosynthetic enzyme N-deacetylase/N-sulfotransferase, which converts GlcNAc into its N-sulfated version GlcNS; then consequently less modified by epimerase activity; and subsequently by
Conclusions
Although highly and ubiquitously abundant in vertebrates (mammals as the main representative organisms), GAGs of unique structure can occur more often in invertebrate animals. The invertebrates can be either from terrestrial habitat like the pulmonate gastropod mollusk A. fulica (giant African snail), which expresses the AS, or from marine habitat like the sea cucumbers, which express FucCSs, the tunicates, which express DSs of unusual sulfation patterns, the bivalve mollusk N. nodosus (lion's
Funding
This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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