Mucins — large extracellular proteins that are heavily glycosylated with complex oligosaccharides — establish a selective molecular barrier at the epithelial surface and engage in morphogenetic signal transduction. Alterations in mucin expression or glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion and immune surveillance. Mucins are used as diagnostic markers in cancer, and are under investigation as therapeutic targets for cancer.

Mucins in cancer: Protection and control of the cell surface

Protein damage and degradation by oxygen radicals. I. general aspects.

Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.

https://www.future-science.com/doi/pdf/10.2144/04375RV01

Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.

Almost all of the key molecules involved in the innate and adaptive immune response are glycoproteins. In the cellular immune system, specific glycoforms are involved in the folding, quality control, and assembly of peptide-loaded major histocompatibility complex (MHC) antigens and the T cell receptor complex. Although some glycopeptide antigens are presented by the MHC, the generation of peptide antigens from glycoproteins may require enzymatic removal of sugars before the protein can be cleaved. Oligosaccharides attached to glycoproteins in the junction between T cells and antigen-presenting cells help to orient binding faces, provide protease protection, and restrict nonspecific lateral protein-protein interactions. In the humoral immune system, all of the immunoglobulins and most of the complement components are glycosylated. Although a major function for sugars is to contribute to the stability of the proteins to which they are attached, specific glycoforms are involved in recognition events. For example, in rheumatoid arthritis, an autoimmune disease, agalactosylated glycoforms of aggregated immunoglobulin G may induce association with the mannose-binding lectin and contribute to the pathology.

http://science.sciencemag.org/content/sci/291/5512/2370.full.pdf

Glycosylation and the immune system

https://www.cell.com/cell/pdf/0092-8674(91)90035-W.pdf

Membrane organization of the dystrophin-glycoprotein complex

Labeling of proteins by reductive methylation using sodium cyanoborohydride.

Why are proteins O-glycosylated?

The effect of carbohydrate on the conformation and chain dimensions of mucous glycoproteins was investigated by using light-scattering and circular dichroism studies of native, asialo, and deglycosylated (apo) ovine submaxillary gland mucin (OSM). OSM is a large glycoprotein that is extensively O-glycosylated by the disaccharide alpha-NeuNAc(2-6)alpha-GalNAc-O-Ser/Thr. Measurements of root mean square radius of gyration, (Rg2)1/2, and hydrodynamic radius, Rh, for OSM and its derivatives were carried out as a function of molecular weight by using static and dynamic light-scattering techniques. The results were fit to the wormlike chain model for describing the dimensions of extended polymer chains. By use of this model, values of h, the length per amino acid residue, and q, the persistence length, which is a measure of chain stiffness, were obtained. These values were then used to assess the conformation and degree of chain extension of intact OSM and its partially and totally deglycosylated derivatives. Native and asialo mucin are found to be highly extended random coils, with asialo mucin having a somewhat less extended structure than intact mucin. Upon the complete removal of the carbohydrate side chains, the extended structure characteristic of intact and asialo mucin collapses to chain dimensions typical of denatured globular proteins. Conformational analyses based on the rotational isomeric state model were also performed by using the probability maps of N-acetyl-O-(GalNAc)-Thr-N-methylamide as starting conformations for native and asialo mucin. The results suggest that both the glycosylated and nonglycosylated residues in native mucin may occupy a small region of conformational space having -90 degrees less than phi less than -60 degrees and 60 degrees less than psi less than 180 degrees, while a slightly broader range is found to fit asialo mucin. The proposed conformations obtained for these mucins are consistent with their circular dichroism spectra. Significantly larger ranges of phi and psi values were obtained for apo mucin, as would be expected from its circular dichroism spectra and increased flexibility. These results indicate the expanded mucin structure is the direct result of peptide core glycosylation. These observations together with the results of earlier studies indicate that steric interactions of the O-linked GalNAc residue with the peptide core are primarily responsible for the expanded mucin structure and that these perturbations extend to the nonglycosylated amino acid residues. This expanded mucin conformation must be a significant determinant of the viscoelastic properties of these molecules in solution.

Neil Jentoft

Papers

Labeling of proteins by reductive methylation using sodium cyanoborohydride.

Why are proteins O-glycosylated?

Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin

[50] Protein labeling by reductive alkylation

13C NMR studies of ribonuclease A methylated with [13C]Formaldehyde.