The British Biophysical Society

The Biophysics of Polysaccharides, Oligosaccharides and Glycosylation: Glycoproteins, Glycolipids and Proteoglycans

Elizabeth Hounsell

 

The majority of proteins are glycosylated i.e. they have attached oligosaccharide chains and are hence termed glycoproteins. The oligosaccharides, also called carbohydrate or sugar because their building blocks are related to glucose, are often quite large (as large as some protein domains for example) and they have many functions in molecular interactions. The building blocks are linked together in different ways in linear or branched chains. Near branch points there is local conformation which can form recognition motifs. Long linear sections can be quite flexible and hence alter the solution properties of the glycoproteins. Their motion and conformation are explored using NMR spectroscopy and molecular modeling. NMR can also be used to unravel the oligosaccharide sequence, the types of building blocks and how they are linked together. Oligosaccharide recognition motifs found on glycoproteins can also be present on mammalian glycolipids and bacterial polysaccharides for example. Carbohydrate chemistry has found new favour recently for designing specific structures as ligands for carbohydrate binding proteins and also other macromolecules, eg in DNA-oligosaccharide interactions in cancer and infection.

Oligosaccharide function

The initial characterisation of specific oligosaccharides as the blood group antigens recognised by serum antibodies (immunoglobulins) has burgeoned into a major area of oligosaccharides as tumour associated and tissue differentiation determinants. (Fig 1). Besides being cell markers with endogenous roles in mammals in cell adhesion and trafficking, microorganisms have often chosen oligosaccharide sequences as "molecular fly paper" to bind to their mammalian hosts for mediation of colonisation and infection. Viruses, bacteria, parasites etc are themselves glycosylated providing other targets for drug intervention. In other pathological states from diabetes to dementia, saccharide derivatives are being researched as possible therapeutics. Given the diversity of structures and functions, the potential is limitless.

Not to be forgotten is the already multimillion-dollar industry using polysaccharides as stabilisers, thickeners and emulsifiers in foods and a wide range of products from cell growth media to cosmetics. Artificial sweeteners and biocompatible surfactants are two new commercial success stories exploiting carbohydrate chemistry. In the pharmaceutical area there are already several near market products based on the rapidly increasing rational understanding of oligosaccharide conformation and biological function (a-d below). Additional promising areas are being explored by venture capital companies (d-f below) and the most potent anti-cancer natural products and important tumour antigens are carbohydrate based (g-h below). Polysaccharides are finding their way into new applications such as wound and ulcer healing and cell transplantation (eg in diabetes). Substituted cyclodextrins are the new inclusion complexes and chiral media. Many marine polysaccharides await exploitation in both the food and medical industries and it can be foreseen that the fields of the future will be engineered to produce polysaccharide crops of desired functionality.

Success stories in the pharmaceutical industry with widening potential in the future are:
a) Computer aided rational drug design and chemical synthesis has brought to market low molecular weight heparin oligosaccharides which mediate anticoagulation without side affects. Interactions of heparin-like glycans are now also being explored in growth control, cancer metastasis and neuropathology (Fig 2).

b) A carbohydrate-based drug to inhibit the neuropathology which leads to senile dementia is being promoted which specifically disrupts the laying down of serum amyloid plaques.

c) A monosaccharide derivative is being heralded as a cure for influenza. Again computer aided drug design and carbohydrate synthesis have gone hand-in-hand to yield an inhibitor of a pathological process, this time essential carbohydrate cleavage by a virus enzyme.

d) In another disease initiated by virus infection, AIDS, naturally occurring and chemically synthesised inhibitors of oligosaccharide biosynthesis are in clinical trials (Fig 3).

e) In addition to viruses, other microorganisms involve the carbohydrates of the host in virulence. Inhibition of infectivity and increasing immunity are two areas being actively researched in bacterial and mycobacterial diseases, from meningitis to intestinal infections and TB.

f) Colonisation by bacteria is mediated by carbohydrate binding proteins with mammalian homologues which bind oligosaccharides to mediate cell-to-cell attachment. The commercial promise is that characterising the lectins in humans and synthesising oligosaccharide mimics, will lead to a new era of anti-inflammatory and anti-metastasis drugs. Preliminary trials in arthritis using sulfated Lewisx derivatives have shown encouraging results.

g) In cancer and xenotransplantation, the cell surface oligosaccharide structures elicit immune responses which can be targets of anti-tumour therapy and new diagnostics and can be manipulated to reduce rejection of foreign transplant organs.

h) Some of the most potent anti-cancer natural products, antibiotics and neurotoxins have oligosaccharide as their key structural motifs. Gradually more and more difficult molecules are being synthesised by the carbohydrate chemist. The future potential is enormous, for the design of carbohydrate-based ligands.

Biosynthesis

The enzymes which catalyse the condensation reaction between monosaccharides are called glycosyltransferases as a family and further specified by the type of glycose, the linkage position and acceptor monosaccharide. The enzyme which transfers a preformed oligosaccharide (of composition Glc3Man9GlcNAc2) from its covalently attached lipid carrier to the nitrogen of the asparagine in the consensus amino acid sequence Asn.Xxx.Thr/Ser of nascent polypeptide chains as they are synthesised on the ribosome is called an oligosaccharyl transferase. The oligosaccharide chains are further processed by a series of glycosidases and glycosyltransferases as the glycoproteins are transported through the endoplasmic reticulum (ER) and the Golgi apparatus. Here other oligosaccharides are built up on GalNAc linked to the oxygen group of Ser or Thr (O-linked protein glycosylation as opposed to N-linked glycosylation above), on Xyl linked to the oxygen of Ser to give proteoglycans and on Glc linked to ceramide (the main family of glycolipids; glycosphingolipids, cerebrosides, gangliosides).

Another major glycosylation mechanism occurs on the cytoplasmic face of intracellular membranes where an enzyme catalyzes the addition of GlcNAc to the oxygen of Ser/Thr and provides a regulatory role reciprocal to phosphorylation in the nucleus and cytoskeleton. N-linked oligosaccharide glycosylation of composition Glc1Man9GlcNAc2 has a role intracellularly at the ribosome as a ligand for the protein calnexin which is involved in the correct folding and trafficking of nascent glycoproteins. Further down the line, after loss of the Glc, glycoproteins which are destined to become lysosomal enzymes are processed by addition of GlcNAc-PO4 to two of the penultimate Man residues of the oligosaccharide chain and the phosphate acts as a signal for transport to the lysosyme.

For other glycoproteins cleavage of the Glc to give high mannose chains can be followed by specific mannosidase activity and the addition of GlcNAc on one arm (hybrid chain) and/or with further mannosidase activity and the addition of GlcNAc on both arms (complex chain). Additional glycosyltransferases catalyse the biosynthesis of the large number of oligosaccharide sequences found. These, as with oligosaccharide extensions to the GalNAc-O-Ser/Thr, Xyl-O-Ser and glycolipid chains, carry out their functions at the cell plasma membrane and on secreted molecules. The cell membrane sees an additional type of saccharide, that present in protein GPI anchors which has a glycan (G) component linked through a mannose- 6-PO4-ethanolamine linkage to the carboxy terminus of some extracellular proteins and to the cell membrane via phosphatidyl inositol (PI). Inositol and its various phosphorylated forms have other roles intracellularly as indispensable signal molecules (another area for carbohydrate chemistry in designer intervention therapy). GPI anchors were originally found in parasites such as trypanosomes but are now being increasingly implicated in different mammalian glycoproteins.

PI links also occur in mycobacteria (eg those causing leprosy and tuberculosis) which have a variety of unique types of glycoconjugates called glycopeptidolipids (previously mycolipids) and trehalose (Glc1-1Glc) - containing lipooligosaccharides (LOS). The major saccharides of bacteria are the lipopolysaccharides (capsular polysaccharides, CPS) composed of repeating di- to hexa- saccharide units which carry the dominant antigenic determinants for host immunity, but can also be mimics of mammalian oligosaccharides (eg see the discussion on blood groups below). Bacteria also have their own glycoproteins, similar but quite distinct from those of mammals, and a range of unique structures involving peptidoglycans (muramic acid and GlcNAc repeating polymer crosslinked to a novel aminoacid framework) and teichoic acids (polymers of sugar, aminoacid and alditol phosphates). Viruses do not encode saccharide processing enzymes in their genome for their own glycosylation although they do express enzymes for processing the host, most famously the neuraminidase of the influenza virus which is of present interest in drug design studies. The coat proteins of viruses are however highly glycosylated by using the host glycosyltransferase machinery and this is one of the mechanisms of avoiding the host immune defence system. Present antiviral strategies (eg in AIDS) therefore include the use of inhibitors of the protein N-glycosylation pathways discussed above which may also reduce viral biosynthesis, infectivity and stability.

Oligosaccharide Conformation

Some of the first oligosaccharides to be characterised were the blood group antigens from the pioneering work of Landsteiner, Morgan, Kabat and Watkins. These were also amongst the first to gain the interest of the synthetic chemists, notably Lemieux who can also be credited with promoting oligosaccharide conformational and recognition studies [Hounsell, 1994]. All this followed on from the elucidation of the structures and stereochemistry of the major monosaccharides (from Emil Fischer onwards) and some of the polysaccharides of plants, bacteria, seaweeds and mammalian food storage, and the identification of carbohydrate-protein complexes from 1880-1940. Early ideas of glycoprotein structure were being formulated in the 1960's including the concepts of Asn- versus Ser/Thr-linked glycosylation. The sialic acid, N- acetylneuraminic acid (NeuAc) was shown to be the monosaccharide cleaved by the 'receptor destroying enzyme' of influenza virus and the terms N and O-linked glycosylation were coined, the latter being described as the oligosaccharides chains of mucins, the high molecular weight glycoproteins which line the gastrointestinal and respiratory tracts [Hounsell et al., 1995].

A lot of the early work on the conformation of carbohydrate molecules was carried out on polysaccharides by X-ray diffraction studies. More recently it has been realised that oligosaccharides can be crystallised and this has opened the way for studies of their interactions which have previously relied on NMR spectroscopy [Hounsell, 1995; Hounsell and Bailey, 1996] and molecular modelling. Good data have also been obtained from X-ray crystallography of complexes of oligosaccharides or glycoproteins with their binding-proteins i.e. anti-carbohydrate antibodies, plant lectins and mammalian carbohydrate binding proteins (CBP) such as the selectins, the galectins, mannose binding protein (a collectin). Data have yet to come on sialoadhesins (the fourth category of endogenous mammalian lectins) but a great deal of data are available from viral (glyco)proteins which interact with sialic acid eg. the influenza virus haemagglutinin and neuraminidase (or sialidase as the neuraminic acid cleaved is one of the family of sialic acids). This latter work has opened up the potentially very profitable area of carbohydrate-based therapeutics. Other areas such as the inhibition of selectin binding in inflammation, proteoglycan interactions in viral infection antibacterial/parasite infection are large potential growth areas.

Reading List

Hounsell, E.F. (1994) Physicochemical analysis of oligosaccharide determinants of glycoproteins. Adv. Carbohydr. Chem. Biochem., 50, 311 - 350.

Barboni E, Rivero B.P, George A.J.T, Martin S.R, Renouf D.V, Hounsell E.F, Barber P.C, and Morris R.J.(1995). The glycophosphatidylinositol anchor affects the conformation of the Thy-l protein. J. Cell Science. 108, 487-497.

Hounsell, E.F. (1995) 1H-NMR in the structural and conformational analysis of oligosaccharides and glycoconjugates. In Progress in NMR Spectroscopy, 27 Eds. J.W. Emsley, J. Feeney and L.H. Sutcliffe, Elsevier p445-474.

Hounsell, E.F., Davies, M.J. and Renouf, D.V. (1996) O-linked protein glycosylation structure and function. Glycoconjugate J. 13,19-26.

Hounsell, E.F. Methods of glycoconjugate analysis. (1997) In H.J. Gabius and S. Gabius Eds Glycoscience: Status and Perspectives Chapman and Hall. pages 15-29

Hounsell, E.F. and Bailey, D. (1997) Approaches to the structure determination of oligosaccharides and glycopeptides using NMR. In Glycopeptides and related compounds: Synthesis, Analysis and Applications. Eds. D.G. Large and C.D. Warren. Marcel Dekker Inc.

Hounsell E. F. (1997) Editor Glycoscience Protocols Methods in Molecular Biology Humana

Hounsell, E. F., and Renouf, D. V., Oligosaccharide epitope diversity and therapeutic potential. (1997) In Glycoimmunology Ed. J. Axford, Plenum Press.

Elizabeth Hounsell is Professor of Biological Chemistry at Birkbeck College. She is Chairman of the Royal Society of Chemistry Carboyhydrate Group, World President of the International Carbohydrate Organisation and Secretary of the European Carbohydrate Organisation. She is Editor of Carbohydrate Reseach and on the Editorial Board of several journals including Biochemical Journal, Molecular Biotechnology, Glycoconjugate Research and Biomedical Chromatography.