Aberrant glycosylation of Igg heavy chain in multiple myeloma.

Although the majority of eukaryotic proteins are glycosylated, there is a dearth of knowledge regarding protein sugar moieties and their changes in disease. Most multiple myeloma cases are characterized by production of monoclonal immunoglobulins (Ig). We studied galactosylation and sialylation of IgG heavy chains in 16 patients with IgG myeloma using lectin blotting and densitometry. In comparison to age and sex matched controls, galactosylation was reduced in multiple myeloma (median 317 vs. 362, range 153-410 vs. 309-447 relative units, p = 0.015, Student's t-test). Sialylation was stage dependent; samples from patients with stage IIA had lowest amounts of sialic acid, IIIA intermediate and IIIB highest (142.6 vs. 185.9 vs. 248.5 relative units, correlation coefficient r = 0.55). Both galactosylation and sialylation levels were independent of age, sex, treatment type, response to treatment, disease duration and IgG and b2 microglobulin concentration. These data indicate that multiple myeloma is characterized by aberrant immunoglobulin glycosylation.

Glycoscience -- a new frontier in rational drug design.

Glycans are the most abundant and most diverse biopolymers in nature. Because of their highly specific interactions with physiological receptors, they participate in many crucial biological processes. All these processes are potential targets for therapeutic intervention, and carbohydrate-based drugs are rapidly being taken up by the modern biotechnology and pharmaceutical industry. Recent developments in the field of glycobiology have overcome the problem of glycan analysis and synthesis; and many compounds based on carbohydrates are now in various stages of clinical trials. This article presents glycoproteins in a new light, as an important biopharmaceutical target, giving an overview of their potential use as therapeutic glycoproteins and proteoglycans, inflammation blockers, cancer therapeutics and vaccines, inhibitors of pathogenic microbes, viral inhibitors and potential aids in the treatment of lysosomal diseases, neurological diseases and transplantation rejection.

Galectin-3: an open-ended story.

Galectins, an ancient lectin family, are characterized by specific binding of beta-galactosides through evolutionary conserved sequence elements of carbohydrate-recognition domain (CRD). A structurally unique member of the family is galectin-3; in addition to the CRD it contains a proline- and glycine-rich N-terminal domain (ND) through which is able to form oligomers. Galectin-3 is widely spread among different types of cells and tissues, found intracellularly in nucleus and cytoplasm or secreted via non-classical pathway outside of cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intra- and extracellular proteins galectin-3 affects numerous biological processes and seems to be involved in different physiological and pathophysiological conditions, such as development, immune reactions, and neoplastic transformation and metastasis. The review attempts to summarize the existing information on structural, biochemical and intriguing functional properties of galectin-3.

Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein-RNA interactions.

The Erm family of adenine-N(6) methyltransferases (MTases) is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Hence, these proteins are important potential drug targets. Despite the availability of the NMR and crystal structures of two members of the family (ErmAM and ErmC', respectively) and extensive studies on the RNA substrate, the substrate-binding site and the amino acids involved in RNA recognition by the Erm MTases remain unknown. It has been proposed that the small C-terminal domain functions as a target-binding module, but this prediction has not been tested experimentally. We have undertaken structure-based mutational analysis of 13 charged or polar residues located on the predicted rRNA-binding surface of ErmC' with the aim to identify the area of protein-RNA interactions. The results of in vivo and in vitro analyses of mutant protein suggest that the key RNA-binding residues are located not in the small domain, but in the large catalytic domain, facing the cleft between the two domains. Based on the mutagenesis data, a preliminary three-dimensional model of ErmC' complexed with the minimal substrate was constructed. The identification of the RNA-binding site of ErmC' may be useful for structure-based design of novel drugs that do not necessarily bind to the cofactor-binding site common to many S-adenosyl-L- methionine-dependent MTases, but specifically block the substrate-binding site of MTases from the Erm family.

Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'.

Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N(6) methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC' (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m(6)A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC', we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m(6)A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m(6)A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family.