Ap Corfield

Heterogeneity and Persistence Length in Human Ocular Mucins

Atomic force microscopy (AFM) has been used to investigate the heterogeneity and flexibility of human ocular mucins and their subunits. We have paid particular attention, in terms of theory and experiment, to the problem of inducing the polymers to assume equilibrium conformations at a surface. Mucins deposited from a buffer containing Ni(2+) ions adopt extended conformations on mica akin to those observed for DNA under similar conditions. The heterogeneity of the intracellular native mucins is evident from a histogram of contour lengths, reflecting, in part, the diversity of mucin gene products expressed. Reduction of the native mucin with dithiothreitol, thereby breaking the S==S bonds between cysteine residues, causes a marked reduction in polymer length. These results reflect the modes of transport and assembly of newly synthesized mucins in vivo. By modifying the worm-like chain model for applicability to two dimensions, we have confirmed that under the conditions employed mucin adsorbs to mica in an equilibrated conformation. The determined persistence length of the native mucin, 36 nm, is consistent with that of an extended, flexible polymer; such characteristics will influence the properties of the gels formed in vivo.

Glycan structures of ocular surface mucins in man, rabbit and dog display species differences

The composition of the mucus gel of the tear film reflects the competing needs for transparency, stability, hydration, and protection of the ocular surface. Mucins form the macromolecular scaffolding of this hydrated gel, and glycans decorating these glycoproteins represent a rich source of binding ligands that may both modulate microbial binding and regulate the physicochemical characteristics of the gel. This study compares the structure of O-linked glycans derived from the ocular mucins of three species, to determine whether the ocular surface microenvironment dictates the need for a common pattern of O-linked carbohydrate structures. Ocular mucus aspirates were collected from healthy humans, rabbits and dogs. Mucins were purified using standard protocols. O-glycans were released by hydrazinoloysis and subsequently analysed by a combination of HPLC, exoglycosidase digestions and LC–MS/MS. A total of 12 different O-glycans were identified. In human ocular mucin, the majority were negatively charged and terminated in sialic acid, whilst those from rabbit or dog were mainly neutral and terminated in α 1-2 fucose and/or α 1-3 N-acetylgalactosamine. The glycans were short: the most common structures being tetra-, tri- or disaccharides. Less elaborate glycan structures are encountered at the ocular surface than at many other mucosal surfaces. Species-specific glycan expression is a feature of ocular surface mucins, and has implications for their defensive properties where different microbial and environmental challenges are encountered.

Atomic force microscopy of the submolecular architecture of hydrated ocular mucins

High-resolution atomic force microscopy has been applied to the imaging of intact human ocular mucins in a near-physiological buffer. The mucins displayed a range of lengths from several hundred nanometers to several microns. By varying the ionic composition of the imaging environment, it was possible to image molecules rigidly fixed to the substrate and the motion of single molecules across the substrate. From static molecular images, high-resolution line profiles show a variation of up to +/-0.75 nm in thickness along the molecule. This variation is localized in regions of several tens of nanometers. It is interpreted in terms of the varying glycosylation along the mucin and is consistent with the known size of oligosaccharides in ocular mucins. The dynamic images indicate the possibility of following mucin interactions in situ.

Human preocular mucins reflect changes in surface physiology

Background/aims: Mucin function is associated with both peptide core and glycosylation characteristics. The authors assessed whether structural alterations occurring during mucin residence in the tear film reflect changes in ocular surface physiology. Methods: Ocular surface mucus was collected from normal volunteers as N-acetyl cysteine (NAcCys) washes or directly from the speculum after cataract surgery. To assess the influence of surface health on mucins, NAcCys washings were also obtained from patients with symptoms, but no clinical signs of dry eye (symptomatics). Mucins were extracted in guanidine hydrochloride (GuHCl) with protease inhibitors. Buoyant density of mucin species, a correlate of glycosylation density, was followed by reactivity with anti-peptide core antibodies. Mucin hydrodynamic volume was assessed by gel filtration on Sepharose CL2B. Results: Surface fluid and mucus contained soluble forms of MUC1, MUC2, MUC4, and MUC5AC and also the same species requiring DTT solubilisation. Reactivity with antibodies to MUC2 and MUC5AC peaked at 1.3–1.5 g/ml in normals, while dominated by underglycosylated forms in symptomatics. Surface mucins were predominantly smaller than intracellular species. MUC2 size distributions were different in symptomatics and normals, while those of MUC5AC were similar in these two groups. Conclusions: A reduction in surface mucin size indicates post-secretory cleavage. Dissimilarities in surface mucin glycosylation and individual MUC size distributions in symptomatics suggest changes in preocular mucin that might precede dry eye signs.

Eukaryotic protein glycosylation: a primer for histochemists and cell biologists

Proteins undergo co- and posttranslational modifications, and their glycosylation is the most frequent and structurally variegated type. Histochemically, the detection of glycan presence has first been performed by stains. The availability of carbohydrate-specific tools (lectins, monoclonal antibodies) has revolutionized glycophenotyping, allowing monitoring of distinct structures. The different types of protein glycosylation in Eukaryotes are described. Following this educational survey, examples where known biological function is related to the glycan structures carried by proteins are given. In particular, mucins and their glycosylation patterns are considered as instructive proof-of-principle case. The tissue and cellular location of glycoprotein biosynthesis and metabolism is reviewed, with attention to new findings in goblet cells. Finally, protein glycosylation in disease is documented, with selected examples, where aberrant glycan expression impacts on normal function to let disease pathology become manifest. The histological applications adopted in these studies are emphasized throughout the text.

Ionic liquids in oligosaccharide synthesis: towards mucin-type glycan probes

The present article provides an overview on mucins and their role in biological processes, while aiming to familiarize readers with the current tools available for the synthesis of structurally defined mucin-type glycan probes including the advantages and potential applications of using ionic liquids in the synthesis of this important class of oligosaccharides. Furthermore, we also highlight recent developments in glycoarray technology that can enable high-sensitivity and high-throughput analysis of this important class of protein-carbohydrate interactions.

What role do mucins have in the development of laryngeal squamous cell carcinoma? A systematic review

Mucins are the dominant component in the protective mucus layer on mucosal surfaces including the larynx. Hence, they are part of the first line of defence against external stimuli including effect of smoking in the larynx. We asked whether existing published evidence supported the hypothesis that alteration in mucins expression/production is related to the laryngeal neoplastic process. The objective of this study is to review published evidence for mucins having an important role in normal laryngeal physiology and the development of laryngeal squamous cell carcinoma (SCC). We aimed to review all available literature on mucins in the larynx in order to develop hypotheses to be tested by future research. Thereby, new potential means of prevention and treatment of laryngeal cancer may be developed. A systematic search of all published literature was conducted. Systematic searches were done in the following databases: AMED, BNI, EMBASE, HMIC, MEDLINE, PsycINFO, CINAHL and HEALTH BUSINESS ELITE from their respective inception up to 11 February 2011. The following keywords were used in combination: mucin, larynx and squamous cell carcinoma. Altogether, 53 studies were identified; 43 studies were excluded following screening of the titles and abstracts. Full text manuscripts for ten studies were obtained for detailed evaluation and five studies were included in this review. No single study fulfilled all relevant criteria. Based on the included studies, we now know that MUC1 is definitely expressed in SCC larynx. However, there is no definitive evidence to suggest that MUC1 and MUC2 are aberrantly expressed in SCC larynx as compared to normal larynx. Further studies using the best available detection technique to detect MUC1, MUC2 and other possible relevant mucins i.e., MUC4 on adequate numbers of normal and SCC specimens are needed to confirm the findings of this review.

Mucins: a dynamic biology

In this highlight, we discuss the multifaceted biology of mucins, where molecular architecture meets function, and especially the collective properties of mucin networks and gels that select adherent bacteria and restrict penetration.

Hyaluronan in dry eye and contact lens wearers

Hyaluronan is a ubiquitous glycosaminoglycan, which in the cornea participates in the organization of the extracellular matrix and spacing of collagen fibrils,1 and modulates the access of macromolecules and enzymes to cells.2 Through fibronectin receptors, hyaluronan influences adhesion and migration.3 Hyaluronan is considered a nonspecific response to trauma, and its levels are depressed by corticosteroid therapy.4

Bacterial invasion of HT29-MTX-E12 monolayers: Effects of human breast milk

AIMnThe supramucosal gel, crucial for gut barrier function, might be compromised in necrotizing enterocolitis (NEC). Breast milk is associated with a reduced incidence of NEC. We compared the effects of human breast milk (BM) versus a neonatal formula, Nutriprem 1 (FF), on adherence, internalisation, and penetration of NEC-associated Escherichia coli through monolayers of mucus producing intestinal cells, HT29-MTX-E12 (E12).nnnMETHODSnE12 cells were grown to confluence on membranes permeable to bacteria. E. coli, reference strain and isolated from a NEC-affected intestine, were cultured in LB broth, labelled with fluorescein and biotinylated. Bacteria were suspended in tissue culture medium (TC) or mixtures of TC with BM or FF and applied to the E12 cultures. Bacterial numbers were assessed by fluorescence. DyLight 650-labelled neutravidin, which cannot cross cell membrane, evaluated extracellular bacteria. Fluorescence of basolateral medium was measured to quantify translocation. Bacterial concentrations were compared using the Mann Whitney U test.nnnRESULTSnAfter 1h exposure, E12 cultures adhered or internalised more NEC-derived bacteria than standard strain E. coli and more suspended in FF than BM (P<0.001). A greater proportion of NEC-derived bacteria internalised when suspended in TC or BM. In FF, the NEC-derived strain internalised least. More translocation occurred in BM incubations compared to FF in the first 1-4h: NEC-E. coli less than the reference strain. After 24h translocated bacterial populations were equal.nnnCONCLUSIONnIn this pilot study, breast milk was associated with relatively less adhesion and internalisation of NEC-associated E. coli to mucus covered E12s compared to formula milk.