Glycoconjugates in Cell Function and Therapeutics
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The “glyco” part (see “Carbohydrate Nomenclature”, “Glycoproteins”, “Glycan-to-Protein Linkages”, “Glycosphingolipids”, “Mucin Biophysics”, and “Proteoglycans”) of proteins and lipids inside the cell, at the cell surface, in the extracellular matrix, and on secreted proteins is made up of oligosaccharides that form a hydrodynamic shell and also have many specific functions in recognition.
In the past this shell has been called the glycocalyx suggesting a coating of cells by glycosylation. Thus the typical O- and N-linked oligosaccharide structures of glycoproteins (“Glycoproteins”; “Glycan-to-Protein Linkages”) can be considered the first signals to be seen in cell-cell interactions and are targets of cell regulation. Most secreted glycoproteins have such typical O- and N-glycosylation, but as reviewed, for example, in Brooks et al. (2002), there are many more unusual glycosylation patterns for which biophysics and function have not been clarified. In addition to the external surface of the mammalian cell and secreted glycoproteins, cytoplasmic proteins are also glycosylated giving a signal reciprocal with protein phosphorylation (see “(Glyco)protein Folding Disorders” and “Glycosphingolipids”) on internal organelles have been implicated in apoptosis, for example.
Particular functions rely on the diversity of oligosaccharides which biosynthetically is of two types: (1) many-enzyme-catalyzed-interconverted monosaccharides that are (2) linked together in different ways all precisely enzymatically controlled. The genes for the enzymes take up a large part of the genome of mammalian, bacterial, and all other organisms (except viruses that use their host glycosylation machinery). This has long been suggestive of their important function, some examples of which are given below in cancer, immunology, infection, and structural integrity.
Aberrant glycosylation occurs in essentially all types of cancer and appears to be an early event such as in control of apoptosis and plays a key role in the induction of invasion and metastasis (Ono and Hakomori 2004). A recent example of the latter is the heightened expression of a sialyltransferase, ST6GalNAcV, which specifically mediates brain metastasis of breast cancers (Bos et al. 2009). The nomenclature here relates to O-linked glycosylation via GalNAc (see “Glycan-to-Protein Linkages”) that can have several mono-disaccharide additions defined as core regions I–VIII (Brooks et al. 2002) and that can be further glycosylated or sialylated, the latter by the addition of one of the family of sialyltransferase (ST) catalyzed sialic acids (in humans N-acetylneuraminic acid) to C-6 (designated by “6” in the enzyme nomenclature), C-4, or C-3. Also the expression of sialyl Lewis x (SLex) on core II glycans is associated with hematogenous metastasis (Ono and Hakomori 2004; Kannagi et al. 2004), and the binding of this glycan to selectins (see “Lectins”) is the mechanism whereby leukocytes extravasate from the blood (McEver et al. 1995). This gives a possible mechanism for involvement of glycosylation patterns in metastasis. In addition to being highly represented in tumors, SLex and related glycosylation are prime candidates as possible therapeutics in many aspects of inflammatory states (Hounsell 2001).
Many of the possible short O-glycosylation patterns have been associated with different primary cancer states, but particularly with breast, gastrointestinal, and colorectal carcinomas. Monoclonal antibodies against many of these have been used in detection and therapy. As suggested by the conformation choices of O-glycosylation described in “Glycan-to-Protein Linkages”, monoclonal antibodies can also be raised against O-glycopeptide epitopes, and these have been associated with cancer (Large and Warren 1997; Haavik et al. 1999) and are potential diagnostic markers (Wandall et al. 2010). At the protein level, significant multivalency is given by clustered O-glycosylation sites on mucins, and the MUC genes define a family of these described in “Mucin Biophysics”. The O- and N-glycosylation of many other normal-size proteins have been implicated, for example, osteopontin (OPN) that exemplifies the interconnectedness of different cell surface components in cancer, as a phosphorylated glycoprotein that binds to cell surface integrins and CD44 in normal tissues and functions as a signal transducer to promote adhesion, motility, and survival (Bellahcene et al. 2008). OPN has been shown to facilitate the growth of established tumors linked to distant sites, but this is dependent on the cell-type expressing OPN and is hence thought to be related to the different cell-specific glycosyltransferase levels mediating the different N- and O-glycosylation patterns observed.
As implicated by the example of OPN, changes in glycosylation that occur in cancer can also alter molecular recognition with the immune system and receptor signaling (Bos et al. 2009) and interaction with the extracellular matrix (see “Proteoglycans”). Carbohydrate-protein interactions are mediated by lectins (as defined in “Lectins”). To choose just one example, C-type (for Ca++-dependent) lectin domains (carbohydrate recognition domains, CRDs) are present on a wide variety of cell surface glycoproteins including DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin; CD209) and DC-SIGNR (DC-SIGN related; CD299) which are two type II transmembrane glycoproteins that possess C-terminal C-type CRDs. In addition to binding endogenous cell adhesion molecules (CAMs), DC-SIGN and DC-SGINR interact with carbohydrate structures on the surfaces of a broad range of lethal infectious reagents. Thus understanding of the interaction can lead to specific therapeutics in infection and inflammation. To this end the CRDs at approximately 16 kDa have been studied with various carbohydrate ligands by X-ray crystallography and NMR (Feinberg et al. 2007; Hibbert et al. 2005). X-ray studies cannot accommodate critical biophysical considerations such as dynamics, temperature, and kinetics in real time; hence NMR and modeling have also been important in elucidating their biophysics (see “Molecular Dynamics Simulations of Carbohydrates”, “Carbohydrate NMR Spectroscopy”, and “X-Ray Diffraction and Crystallography of Oligosaccharides and Polysaccharides”).
C-type lectins involved in immune regulation are described as selectins on lymphocytes (L-selectin), endothelial cells (E-selectin), and platelets (P-selectin) from the cells that they were first observed on, but this is an oversimplification as is what follows: lymphocytes in the blood stream react to bacterial signal molecules and upregulate their L-selectin. Meanwhile, the endothelial cells near the site of infection upregulate E-selectin as well as the carbohydrate ligand for L-selectin which is called P-selectin ligand 1 (PSGL-1). Lymphocyte traffic is thus slowed down enough to bind to the endothelial cells (a procedure called “rolling”), and then integrins tighten their hold so that the lymphocytes traverse (extravasate) across the vascular endothelium to the site of infection. Various places along the line are targets for therapeutic intervention. Reference to the Leucocyte Handbook (1993, 1997) that defines the CD nomenclature referred to above also suggests roles of oligosaccharides in the overall structure of the cell surface biophysics as, for example, molecular spacers for helping coordinate the interactions of multiple glycoproteins involved in T-cell recognition of peptide epitopes presented on MHC glycoproteins of B and dendritic cells.
Bacteria synthesize many glycoproteins, lipopolysaccharides (LPS), and polysaccharides (PS). LPS and PS are reviewed elsewhere (“Bacterial Lipopolysaccharide, OPS, and Lipid A”; “Bacterial Polysaccharide Structure and Biosynthesis”). In addition bacterial protein glycosylation (“Glycan-to-Protein Linkages”) is described further in a good short review in Brooks et al. (2002). Among their oligosaccharide diversity are represented many of the carbohydrate epitopes also synthesized by humans, and this, for example, is thought to be the mechanism for our blood groups and other cross-reactivities involved in human pathology such as the Guillain-Barre syndrome. Human blood group oligosaccharide structures are shown in the entry on “Lectins”. In short, people who are blood group A express an enzyme that catalyzes the addition of GalNAc to a backbone Gal1,3/4[Fuc1,2]GlcNAc structure called H. People who are blood group B express an enzyme that catalyzes the addition of Gal to H. Blood group A serotypes have circulating antibodies to blood group B antigen and vice versa because bacteria also synthesize the blood group A and B structures to which we make antibodies. However, immune tolerance mechanisms are bought into play so that those with blood group B antigens do not have antibodies to blood group B and those with blood group A antigens do not have antibodies to blood group A. During blood transfusion, if blood group B erythrocytes are given to blood group A people, or vice versa, the erythrocyte-antibody interaction causes hemagglutination. Blood group O, by the way, is the absence of the enzymes that catalyze either of the A and B antigens; hence this serotype has antibodies to both A and B and can only accept blood from other O-serotypes, but can donate their red blood cells to either A or B serotypes (a universal donor).
The lectins initially used in blood typing were initially found in plants but since then have been found to have many uses as reagents in mammalian biomedicine. However, the function of their binding to their natural ligands in plants is still not known, but is presumed to have some function in the “medical cabinet” of plants for reagents against viral, bacterial, and fungal pathogenesis. In addition there is a huge diversity of plant natural products having many “Glycosylated Natural Products” that also function in Nature’s medicine cabinet, one that we have amply raided for our own therapeutics. Plants are normally associated with the polysaccharides such as “Pectin Biophysics” that are involved in structure rather than recognition.
Viruses use the glycosylation machinery of the host cell that they are infecting in order to add oligosaccharides to their external proteins. High mannose N-glycans on viruses in particular have been targets for therapeutic intervention in order to destabilize virus structure (e.g., HIV) and to inhibit, for example, the multiple modes of binding that enhance the affinity of DC-SIGN mentioned above (Feinberg et al. 2007). It appears that changes in N-glycosylation pattern via changes at the genome level moving the N-glycosylation sequon AsnXxxSer/Thr (“Glycan-to-Protein Linkages”) are also one of the mechanisms that helps the virus evade the immune system which is targeted at exposed protein epitopes (any carbohydrate antigens being also those of the host and hence tolerated). Detailed epitope mapping on, for example, the highly glycosylated gp120 of HIV and the hemagglutinin of influenza virus has identified protein epitopes and mapped changing glycosylation patterns.
The mention of the word hemagglutinin of influenza virus alerts us to the fact that viruses also use the host glycosylation in their infectivity mechanisms. In short, the influenza virus has two glycoproteins on its surface: the hemagglutinin that binds to host cell NeuAc2,6 Gal at the end of N- and O-linked chains of the host cell surface glycoproteins of the epithelia of the human respiratory tract and a neuraminidase that cleaves NeuAc2,6 Gal bonds. Once the virus is bound, the cell is infected, more virus is synthesized, and this adheres to the cell surface until the neuraminidase cleaves the cell surface NeuAc2,6Gal bond releasing the virus to infect another cell. Newcastle disease virus, Sendai virus, and fowl plague virus similarly have a hemagglutinin-neuraminidase system, and polyomaviruses and coronaviruses also bind sialic acids. Other viruses may use sulfated glycoproteins as a mechanism of infectivity. As can be imagined, much effort has gone into characterizing at the biophysical level the specific interactions for the rational drug design of inhibitors, the most successful of which are Relenza and Tamiflu.
There is an awe-inspiring diversity of the structure and mechanisms involved in glycoconjugate interactions that are only beginning to be explored as therapeutics. Inhibition of carbohydrate-protein interactions is a difficult area as many of the individual interactions are of relatively low affinity, but techniques to induce multivalency are being exploited. Elucidating the biophysics of the enzymes involved has shown considerable promise.
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