Preparation and properties of porcine submaxillary mucins

doi:10.1016/0003-9861(69)90148-9

Archives of Biochemistry and Biophysics

Volume 129, Issue 1, January 1969, Pages 49-56

https://doi.org/10.1016/0003-9861(69)90148-9 Get rights and content

Abstract

An improved method for the preparation of porcine submaxillary mucin has been developed. As with bovine and ovine submaxillary mucin, two products, a major and minor mucin, have been reproducibly resolved from pooled porcine submaxillary glands by a final purification step involving treatment with hydroxylapatite gel. Tiselius electrophoresis, ultracentrifugation, disc electrophoresis, immunoelectrophoresis, and constancy of chemical composition provided evidence for the absence of contamination by extraneous proteins. Both mucins were readily soluble in water, gave extremely viscous solutions, and had similar compositions that closely resembled that of a single mucin fraction isolated earlier in these laboratories. However their compositions differ markedly from those of two porcine submaxillary glycoproteins described by Katzman and Eylar (3).

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In fact, these two mucin variants are even commercially available and used by many scientists in various research fields [72–74]. Yet, in addition to serving as source for PGM and BSM, porcine and bovine mucus has also been sampled from other mucosal systems [75–77]. Other mammals, from which mucins have been purified, include rats and mice [46,78–83], sheep [71,84,85], rabbits [86,87], dogs [87–89], horses [90], and monkeys [91].
One of the main challenges in the field of drug delivery remains the development of strategies to efficiently transport pharmaceuticals across mucus barriers, which regulate the passage and retention of molecules and particles in all luminal spaces of the body. A thorough understanding of the molecular mechanisms, which govern such selective permeability, is key for achieving efficient translocation of drugs and drug carriers. For this purpose, model systems based on purified mucins can contribute valuable information. In this review, we summarize advances that were made in the field of drug delivery research with such mucin-based model systems: First, we give an overview of mucin purification procedures and discuss the suitability of model systems reconstituted from purified mucins to mimic native mucus. Then, we summarize techniques to study mucin binding. Finally, we highlight approaches that made use of mucins as building blocks for drug delivery platforms or employ mucins as active compounds.
A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase
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Thus, we revealed that PSG had two types of mucins showing different glycan profiles. It has been reported that PSM has two types of mucins with slightly different monosaccharide compositions [20,21]. Savage et al. reported that O-linked glycans from PSM are a mixture of NeuAc α2-3 Gal β1-3 GalNAc (3), NeuGc α2-3 Gal β1-3 GalNAc (5), Gal β1-3(NeuAc α2-6) GalNAc (2), and Gal β1-3(NeuGc α2-6) GalNAc (4) at a molar ratio of 69:22:4:5 [22].
O-Linked glycan liberation from proteins through reductive beta-elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan analysis in glycan biomarker research and microheterogeneity evaluation of biopharmaceutical glycosylation. Here, we introduce an alternative method by using hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and optimize the reaction conditions. The developed method afforded comparable results to those of hydrazinolysis, but with less degraded products. In addition, we examined the compatibility of the optimized O-linked glycan liberation with denaturant and detergents. The optimized method also released glycans containing NeuGc without degradation or deacylation. To demonstrate the feasibility of the developed method, we analyzed O-linked glycans of porcine submaxillary mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. The method for O-linked glycan liberation and fluorescent labeling presented here was easy and rapid, and will be practically useful for O-linked glycan analyses.
Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes
2001, European Journal of Cell Biology
An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b₅ is discussed.
Effect of lipids on the structure and rheology of gels formed by canine submaxillary mucin
1997, Biorheology
Rheological experiments have shown that canine submaxillary mucin (CSM) forms gels in aqueous solution at low ionic strength and in 6M GdnHCl. Examination of specimens of intact CSM and also its subunits prepared by reduction and carboxymethylation showed that the presence of lipid increases the gel-forming capability, probably as a result of enhancement of the intermolecular hydrophobic interactions. The rheological evidence for gelation is that substantially larger values of the oscillatory storage modulus, G'(ω), and the dynamic complex viscosity, η^*(ω), are observed for lipid-containing CSM. TMs is backed up by electron micrographs of freeze fractured specimens, where we observe a network morphology in which the cross-links are formed as a result of non-bonded interactions between a number of CSM chains. The intermolecular interactions responsible for gelation probably involve hydrophobic association between the interdigitated oligosaccharides, and/or between the non-glycosylated regions of the protein core, and can occur even in a highly chaotropic medium (6M GdnHCl). In contrast to previous experiments with porcine submaxillary mucin and human tracheobronchial mucin, which form microphase-separated gels in aqueous solution, CSM solutions undergo macroscopic phase separation into polymerrich (gel) and polymer-poor (sol) phases. These data point to stronger hydrophobic interactions in lipid-containing CSM.
Rheological studies of the interaction of mucins with alginate and polyacrylate
1996, Carbohydrate Research
The associative interaction of purified ovine and porcine submaxillary mucins (OSM and PSM) with sodium alginate was evaluated by comparing the rheological properties of mixtures against those of pure alginate and mucin in dilute, semi-dilute, and concentrated solutions. These systems were investigated as models for the interaction of mucin with the extracellular alginate produced by Pseudomonas aeruginosa. In dilute solution, evidence for such interaction cannot be obtained because aggregate species exist both in the OSM-alginate mixtures as well as in pure OSM. However, in the semi-dilute regime, mixtures containing a higher proportion of mucin show systematically higher viscosities than those predicted by simple additivity. In concentrated solutions containing higher proportion of mucin, an enhanced elastic response is observed. These results demonstrate a substantial binding interaction of mucins with alginate. This property is not observed in mixtures containing a high proportion of alginate, suggesting that mucins possess relatively low numbers of interacting sites. Introduction of 3 mM Ca²⁺ ions to all mucin-alginate mixtures enhances the elasticity due to gelation of alginate. Finally, comparison of the rheological properties of PSM-alginate mixtures with those of PSM-polyacrylate mixtures indicates that the binding strength of alginate to mucin is significantly weaker than that of polyacrylate.
Solvent effects on the viscoelastic behavior of porcine submaxillary mucin
1995, Biorheology
Rheological methods have been used to investigate the intermolecular interactions of porcine submaxillary mucins (PSM) in solution. PSM is a high molecular weight glycoprotein consisting of a linear, semi-flexible protein backbone to which a large number of oligosaccharides (1–5 saccharide units) are attached as side chains. Concentrated aqueous solutions of PSM containing different amounts of guanidine hydrochloride (GdnHCl) were subjected to both controlled stress and controlled strain rheological analyses. In the absence of GdnHCl, PSM solutions exhibit viscoelastic properties characteristic of a gel: the storage modulus, G′, is much larger than the loss modulus, G″, at all deformation frequencies, and the compliance is 100% recoverable at small stresses, indicative of strong intermolecular interactions. In 3.0 M aqueous GdnHCl, PSM forms a viscoelastic solution, with G″ > G′ at all frequencies and a relatively small recoverable compliance, pointing to disruption of the intermolecular interactions by the chaotropic salt. Intermediate behavior is observed in 1.5 M GdnHCl, characteristic of a marginal gel: G′ ≈ G″ and greater than 50% recoverable compliance. In dilute solution, PSM behaves viscoelastically as a typical polyelectrolyte. However, concentrated solutions are turbid, the turbidity decreasing as GdnHCl is added, indicating that extensive intermolecular association accompanies the gelation process. The results show that although PSM is secreted in nature as a viscous solution, it can form gels that are similar to those of tracheobronchial and gastric mucins, and suggest common features to the gelation mechanism, with the strength of the gel correlated with the length of the oligosaccharide side chains.

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^☆: This work has been supported by a grant from the National Institutes of Health (AM-04619) to Dr. Ward Pigman, and by a grant from the National Cystic Fibrosis Foundation to Drs. I. L. Schwartz, E. A. Popenoe, and M. de Salegui.

²: Present address: Department of Physiology, the Mount Sinai School of Medicine, New York, New York 10029. This is the address to which inquiries and requests for reprints should be directed.

³: Present address: Department of Physical Medicine, College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032.

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Preparation and properties of porcine submaxillary mucins☆

Abstract

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