Normal Mucin Research Articles

Noninvasive colorectal cancer screening.

Abnormal mucin with the STn epitope is produced by colorectal cancer cells and is immunologically distinguishable from normal colonic mucin. Herein we describe a technique of detecting abnormal mucin in fecal samples as a method of screening for colorectal neoplasia. Soluble glycoproteins from fecal samples of patients with symptoms of bowel disease and asymptomatic volunteer subjects were isolated by centrifugation and ethanol precipitation. The protein content of the soluble fraction was normalized and tested by immunoassay (slot dot). Anti-COTA monoclonal antibody SP-21, which reacts with STn epitope, was applied in the reaction, and the optical density of each slot dot was determined by imaging densitometer. Quantitative values of samples were determined from the standard curve obtained with highly purified COTA. COTA values > 15 microg/ml were considered positive for neoplasia. Results indicated that 5/6 colon cancers, 6/22 adenomas, 1/8 colitis cases, and 2/58 normal patients were positive in the test. The pilot study revealed that COTA assay is sensitive and more specific than Hemoccult screening for colorectal neoplasia.

Identification and Functional Characterization of a Human GalNAc α2,6-Sialyltransferase with Altered Expression in Breast Cancer

We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers. Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis. A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc alpha2,6-sialyltransferase with Gal beta 1,3 GalNAc-O-Ser/Thr and (+/- Neu5Ac alpha 2,3) Gal beta 1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is nonrandomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines. Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant glycosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.

Human conjunctival epithelium in culture: a tool to assay new therapeutic strategies for dry eye.

Conjunctival changes in dry eye syndrome involve alteration in the mucous layer of the tear film. Mucus production and secretion is one of the principal roles of the conjunctival epithelium. Mucins, the main components of the mucous layer, are glycoproteins produced by goblet and non-goblet epithelial cells from the conjunctiva1–3 and by corneal epithelial cells.4 Any process that interferes with normal mucin production also interferes with proper wetting of the ocular surface.

Smoke-induced inhalation injury: effects of retinoic acid and antisense oligodeoxynucleotide on stability and differentiated state of the mucociliary epithelium.

Rabbit tracheal explants, exposed to burning pine wood smoke, were cultured in a chemically defined medium with and without retinoic acid (+/- RA). Exposures of 15-20 minute led to RA-independent degeneration of the mucociliary epithelial sheath. In 10 minute exposures tissue integrity was retained, but epithelial morphology changed from normal pseudostratified columnar to the flattened appearance typical of the squamous phenotype. Despite the dramatic shift in morphology, explants exhibited normal RA-dependent mucin gene expression characteristic of the mucociliary phenotype. Furthermore, electron micrographs showed continued presence of both secretory granules and cilia. RA(+) cultures also showed a normal pattern of adherent epithelial cells. In RA(-) cultures, however, there were prominent intercellular spaces indicating an RA dependence for maintaining adhesive contacts following smoke exposure. An 18-mer mucin antisense oligomer that suppressed mucin gene expression also unexpectedly blocked the smoke induced metaplasia in RA(+) cultures, but the sense oligomer had no effect.

Epitope mapping of anti-MUC1 mucin protein core monoclonal antibodies.

A panel of 56 murine monoclonal antibodies against the MUC1 mucin was analysed for reactivity against purified MUC1 mucin and synthetic MUC1 mucin protein core-related peptides. In an ELISA assay, with purified normal urinary mucin as the target antigen, 48/56 (86%) of antibodies showed positive reactivity. A smaller proportion of antibodies, 31/56 (55%), reacted with a bovine serum albumin conjugate containing the synthetic peptide, APDTRPAPG. This peptide represents the hydrophilic region of the tandem repeat sequence of the MUC1 mucin core. These 31 anti-mucin core antibodies were then evaluated for reactivity with a set of overlapping heptamers with sequences based upon that of the 20-amino acid tandem repeat of the MUC1 protein core. For this purpose, the 'Pepscan' procedure was employed to detect antibody binding to peptides tethered at their carboxy termini to the polypropylene pins. Synthetic peptides binding to these antibodies were identified for 29 of the samples provided and epitopes or 'minimum binding units' were deduced for these. Most antibodies bound to epitopes of 4 amino acid residues with the sequence RPAP occurring most frequently. The results confirm that the short hydrophilic region PDTRPAP in the MUC1 mucin core is immunodominant in the induction of antibodies to the MUC1 mucin since half of the antibodies submitted reacted with this peptide.

Correlative histochemical study providing evidence for the dual nature of human colorectal cancer mucin.

Luminal secretions within colorectal cancers have been assumed to be the counterpart of normal goblet cell mucin. The aim of this study was to establish whether secretory material within colorectal cancers may in fact be traced to different lineages: goblet cells and columnar cells. The distribution of the apomucins MUC1 and MUC2, non-O-acetylated sialic acid and the carbohydrate structures sialosyl Tn, Tn, Lewis(x), sialosyl Lewis(x) and Lewis(y) was studied in normal colorectal mucosa and colorectal cancer specimens using standard histochemical techniques. Unmasking of MUC1 and MUC2 was achieved using periodic acid and saponification-neuraminidase-periodic acid pretreatment respectively. Within normal and malignant epithelium, correlations and/or co-localization could be demonstrated for goblet cells with MUC2, non-O-acetylated sialic acid, sialosyl Tn, Tn (Golgi region) and sialosyl Lewis(x), and for columnar cells with MUC1, Lewis(x), sialosyl Le(x), Tn (cytoplasm) and Lewis(y) (UEA-1). The goblet cell spectrum was associated with mucin-like (type I) luminal secretions within cancers, whereas the columnar cell spectrum characterized non-mucin-like (type II) secretions and intracytoplasmic lumina. These data indicate that colorectal cancer mucin can be broadly separated into two types: secretory mucin linked to cells of goblet lineage and up-regulated membrane-associated mucin of presumed columnar cell origin.

Graft-versus-host disease after small bowel transplantation is associated with host colonic injury.

The present studies were undertaken to evaluate the histologic effects of graft-versus-host disease on the host colon after small bowel transplantation. Graft-versus-host disease was produced in six Lewis x Brown Norway F1 rats by performing vascularized, out-of-continuity small bowel transplants from parental Lewis donors. Host proximal and distal colon were sampled 14 days after operation when signs of graft-versus-host disease, including weight loss and splenomegaly, were present. Tissue was assessed histologically by blinded observer and compared to eight sham-operated controls. Three histologic features were noted to be statistically increased in diseased animals: (1) mucin loss; (2) crypt abscesses; and (3) large lymphoid aggregates in the mucosa and submucosa. These features were more commonly noted in the distal rather than the proximal colon. Another group of five grafted animals treated with cyclosporine A (10 mg/kg/day intramuscularly) still lost weight but did not display overt signs of graft-versus-host disease and had normal-sized spleens. There was normal mucin content and no evidence of crypt abscesses in these treated animals, although large lymphoid aggregates were present. It is concluded that mucin loss, crypt abscesses, and large lymphoid aggregates are characteristics of graft-versus-host disease-induced colonic injury in this model and that these changes are most evident in the distal colon. Cyclosporine A therapy does not completely reverse the histological changes of colonic graft-versus-host disease. This model may be useful in studying the mechanisms by which immune mediated colitides preferentially affect the distal colon.

Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin.

This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.

Direct demonstration of increased expression of Thomsen-Friedenreich (TF) antigen in colonic adenocarcinoma and ulcerative colitis mucin and its concealment in normal mucin.

Increased binding of the lectin peanut agglutinin is a common feature in epithelial malignancy and hyperplasia. This may have considerable functional importance in the intestine by allowing interaction between the epithelium and mitogenic lectins of dietary or microbial origin. Peanut agglutinin binds the disaccharide Thomsen-Friedenreich (TF, T or core 1) blood group antigen, Gal beta (1-3) GalNAc alpha-, but is not totally specific for this site. Consequently, there has been controversy about the presence of this structure in colon cancer; studies with anti-TF monoclonal antibodies have failed to detect it. We have examined the presence of TF antigen in colonic mucus glycoprotein (mucin) using endo-alpha-N-acetylgalactosaminidase (O-Glycanase), which specifically catalyzes the hydrolysis of TF antigen from glycoconjugates. Samples of adenocarcinoma, inflammatory bowel disease (ulcerative colitis), and normal mucin were treated with O-glycanase, the liberated disaccharide was separated from the glycoprotein and analyzed using dual CarboPac PA-100 column high performance anion-exchange chromatography coupled with pulsed amperometric detection. O-Glycanase treatment released increased amounts of TF antigen from both colonic adenocarcinoma (8.0 +/- 3.9 ng/micrograms protein, n = 11; P < 0.0001 ANOVA) and ulcerative colitis mucin (3.3 +/- 0.3 ng/micrograms protein, n = 5; P = 0.04) compared with mucin samples from histologically normal mucosa distant from carcinoma (1.5 +/- 1.1 ng/micrograms protein, n = 9). However, after mild acid treatment to remove sialic acids and fucose, releasable TF antigen was increased in all nine of these histologically normal mucin samples (5.5 +/- 2.6 ng/micrograms protein, P < 0.0002). We conclude that TF antigen is an oncofetal antigen which is expressed in colon cancer, but is concealed by further glycosylation (sialylation and/or fucosylation) in the normal colonic mucosa.

Gastropathies in the Lundehund

The mucin profiles of the gastric mucosa in Lundehunds suffering from intestinal lymphangiectasia were examined and compared to the mucin profiles in control dogs from other breeds. A previous study performed on this material had shown that all examined Lundehunds had gastritis and about 30% had gastric carcinoma. Neutral and acid mucins were identified using the periodic acid‐Schiff (PAS) and Alcian blue (pH 2.5) periodic acid‐Schiff (AB‐PAS) methods. The acid mucins were divided into sialomucins and sulfomucins based on their reaction with high‐iron diamine Alcian blue, pH 2.5 (HID‐AB). In Lundehunds with chronic atrophic gastritis in the fundic and body regions the surface and foveolar epithelium showed a predominantly normal mucin profile although some Lundehunds had a reduced mucin content. The mucous neck cells extended from below the gastric foveolae towards the muscularis mucosae. Morphometric examination showed that the abnormal presence of mucous neck cells occupied 41% of the height of the gastric mucosa in Lundehunds compared to only 19% in the control dogs (p&lt;0.05). Of the four Lundehunds with gastric carcinoma, two possessed neoplastic cells that contained minimal or no mucins. The amount and type of mucin in the neoplastic cells of the remaining two Lundehunds varied both between individuals and within a neoplasm. This study shows that the abnormal presence of mucous neck cells and the associated pseudopyloric metaplasia comprised the predominant changes in the gastric mucin profiles of Lundehunds.

The roles of enteric bacterial sialidase, sialateO-acetyl esterase and glycosulfatase in the degradation of human colonic mucin

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or more O-acetyl esters at positions C7-C9 on the sialic acids retarded the rate of hydrolysis. A specific sialate O-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and a KM of approximately 1 mM sialate O-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[3H]ol 6-O-sulfate with a pH optimum of pH 5.0 and a KM of approximately 1 mM. Faecal extracts from ulcerative colitis (UC) patients had higher sialate O-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged. Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acid O-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects.

Biopathologic characteristics of DNA content in crypt cells of transitional mucosa adjacent to carcinomas of the rectum and rectosigmoid.

The transitional mucosa (TM) adjacent to carcinomas of the large bowel shows histologic and mucin histochemical changes that may indicate premalignant change and may be of prognostic value after radical resection. In this study, 10 anterior resection specimens from patients with carcinomas of the rectum and rectosigmoid were used to compare the nuclear DNA content in TM with those in cancer tissue and with those in nontransitional mucosa (N-TM; i.e., uninvolved mucosa remote from tumors showing normal histologic and mucin histochemical features). The nuclear DNA content was assessed using DNA image cytometry on Feulgen-stained sections. As compared with N-TM, crypts in TM contained greater numbers of cells, were elongated, and were more likely to be branched with marked sialomucin secretion, accompanied by a marked reduction in the normal sulfomucin content. The mean nuclear DNA content in the upper, middle, and lower thirds of crypts was significantly higher in TM than in N-TM, and the nuclear DNA content in TM exhibited no correlation to that in tumors. The results suggest that TM adjacent to carcinomas of the rectum and rectosigmoid has higher proliferative activity, providing further evidence that TM may be an unstable premalignant change.

LONG-TERM FUNCTION OF FREE JEJUNUM TRANSPLANTED FOR ESOPHAGEAL RECONSTRUCTION AFTER RESECTION OF THREE CASES OF A THORACIC ESOPHAGEAL CANCER

Esophageal reconstruction after resection of a thoracic esophageal cancer using free jejunal transfer was performed in 3 cases, and no remarkable postoperative complications were noted in a long term follow-up for 9 months to 4.5 years. The freely transplanted jejuna were found to be well dilatated in postoperative fuluroscopic studies. Strong peristaltic waves and constriction on swallowing occurred 3 months after the operation, but disappeared within 6 months. After that, the passage of foods through the transplanted jejunum was favorable. A positive pressuer zone which moved from the oral to anal site of the transplanted jejunum was recognized in the manometric study. Intermittent interdigestive electric complex were digestive irregular superposed electric complex were observed on electromyography 4 months and 2.5 years after the operation. Normal mucin was observed on the mucosa obtained by the endosocpic biopsy.

Aberrant expression of intestinal mucin antigens associated with colorectal carcinoma defined by a panel of monoclonal antibodies

Small intestine mucin antigen (SIMA) is an oncofoetal antigen for the colon and is distinct from the normal large intestinal mucin antigen (LIMA). In the present study, a panel of anti-SIMA and anti-LIMA monoclonal antibodies (MAb) was used to charaterise altered mucin expression in colorectal adenocarcinomas, by immunohistochemistry and quantitative immunoassays of tissue extracts. These results are compared with CEA expression and correlated with various clinicopathological indices. All mucin MAb reacted with a high proportion of the 100 colon cancers of every stage, histological type (including non-mucinous cancers), differentiation, site, or size. Inappropriate SIMA production was detected by either anti-SIMA MAb 4D3 or 4A1, even in 85% of early stage cancers. MAb 4D3 reacted with a higher proportion of cancers of smaller size and better differentiation. At the subcellular level, both anti-SIMA MAb showed reactivity typical of normal mucin, i.e., goblet cell and extracellular mucin. The normal colonic antigen, LIMA, was also detectable in the majority of cases, but quantitatively overproduced in some cases and reduced in others. However, in contrast to SIMA, LIMA was detected in predominantly undifferentiated cancer cells but not in goblet cells. Heterogeneity of MAb reactivity between cases and complementarity within each cancer was frequently observed. Mucin reactive with at least one of the MAb was detected in all of the CEA-negative cancers. A high rate of inappropriate SIMA expression was also detected in the perineoplastic transitional mucosa (88%, c.f. CEA, 35%) and adjacent, morphologically normal mucosa (80% c.f. CEA, 24%), indicating biochemical changes similar to the cancer. This panel of anti-mucin MAb demonstrated altered mucin glycoprotein metabolism associated with the development and progression of most colorectal cancers, which emphasises their utility as indicators of neoplastic change in the colon, and their superiority to CEA.

Oncofetal expression of the human intestinal mucin glycoprotein antigens in gastrointestinal epithelium defined by monoclonal antibodies

A mucin preparation from a colonic adenocarcinoma was used to prepare monoclonal antibodies (MAbs) that reacted specifically either with normal adult small-intestine mucin antigen(s) (SIMA), or normal adult large-intestine mucin antigen(s) (LIMA). Both SIMA and LIMA show a unique oncofetal pattern of expression. Thus SIMA was expressed in early fetal stomach, large and small intestines but thereafter only in the normal small intestine. SIMA expression was detected immunohistochemically in cancers of the colorectum (82/112) and stomach (48/86). LIMA was detected in the stomach of the early fetus but thereafter only in the normal large intestine. LIMA expression was detected in 61/86 cancers of the stomach. Moreover, both SIMA and LIMA were expressed inappropriately in mucosa adjacent to tumors, indicative of the detection of possible pre-malignant epithelium. We used a sandwich ELISA and biochemical procedures to show that the SIMA and LIMA molecules were large extensively glycosylated multi-unit mucin glycoproteins that differed markedly from each other. SIMA, whether extracted from normal small-intestine or colonic cancers, had a molecular weight above 1.000 kDa, a mean buoyant density 1.33 g/ml and s value of 4.8. LIMA had a molecular weight above 10.000 kDa, a mean buoyant density 1.45 g/ml and an s value 9.5. The SIMA and LIMA epitopes were judged to be carbohydrate in nature by reason of their resistance to harsh physical chemical treatments or protease digestion, and sensitivity to periodate oxidation, neuraminidase or beta elimination. Only the SIMA epitope was sensitive to neuraminidase. In conclusion, MAbs to carbohydrate-dependent epitopes on SIMA and LIMA identify the oncofetal pattern of expression of these distinct intestinal mucin glycoproteins in colonic and gastric carcinoma. These MAbs will be useful in further studies of the significance of oncofetal mucin expression during carcinogenesis.

Localization of Endogenous &amp;beta;-Galactoside-Binding Lectin in Human Cells and Tissues

A rabbit antiserum raised against the 14.5-kilodalton (kDa) subunit of human splenic galaptin was used to probe protein blots of several tissue extracts. For all tissues examined, the only immunoreactive species detected was a 14.5-kDa polypeptide. This antiserum and a rabbit antiserum raised against native lung galaptin were used in immunohistochemical assays to determine the localization of galaptin in selected tissues and cells. In normal colon, galaptin was found prominently in the basement membrane and in the stroma. The cytoplasm of epithelial cells stained lightly for galaptin whereas mucous granules and secreted mucin were uniformly negative for galaptin. Hemagglutination inhibition assays also failed to demonstrate an interaction between galaptin and mucin. Macrophages stained conspicuously for galaptin in colonic and cutaneous tissue as did some capillary walls. In cutaneous tissue, the extracellular matrix and hair follicle cells contained abundant galaptin. Galaptin was absent in basal cell carcinoma and associated stroma. Galaptin was found throughout the cytoplasm of carcinoma cells of gynecologic origin present in effusions. Protein blot analysis of extracts of extracellular matrix synthesized in vitro by endothelial cells confirmed the presence of galaptin in matrix. The results show that: (1) galaptin is variably expressed by different cells and tissues; (2) its cellular location is not restricted to the cell surface; (3) galaptin is not associated with normal mucin; (4) the extracellular matrix is a major site of galaptin deposition, and (5) some malignant tissue may be characterized by a deficiency of galaptin.

Rheological Characterization of Tear Substitutes

AbstractThe steady state viscosity as a function of shear rate was determined for eight commerical tear substitutes and compared with data for high molecular weight sodium hyaluronate solutions. The Zero shear viscosity at steady state varied more than 100-fold from about 2 to 300 cP. Many samples were Newtonian, while some samples exhibited a varying degree of shear thinning (psedudoplastic) behaviour. The results are discussed in realation to the rheological behaviour of normal tears and mucin.

A core protein epitope of the polymorphic epithelial mucin detected by the monoclonal antibody SM‐3 is selectively exposed in a range of primary carcinomas

The monoclonal antibody (MAb) SM-3, which was raised to chemically deglycosylated milk mucin, reacts with an epitope present on the core protein of this mucin which we have referred to as PEM (polymorphic epithelial mucin). Although this mucin is abundantly expressed by both the lactating breast and breast carcinomas, the antibody SM-3 shows very little or no reactivity on the former but does react with 92% of breast carcinomas. Furthermore, SM-3 stains primary carcinomas of the lung, colon and ovary, but on the corresponding normal tissue the epitope is expressed at a much reduced level or not at all. These results indicate that an epitope masked in the normal mucin is exposed in the mucin produced by tumour cells, perhaps due to aberrant glycosylation. An extensive immunohistochemical study of other normal tissues reveals that the majority show only weak focal staining with SM-3 or none at all, the distal tubules and collecting ducts of the kidney, and sebaceous glands being the only normal tissues studied to show homogeneously positive staining.

Physical properties of purified human respiratory mucus glycoproteins: effects of sodium chloride concentration on the aggregation properties and shape.

The biophysical properties of purified native (nonreduced) mucus glycoproteins (mucins) isolated from lung mucus secretions of cystic fibrosis (CF) patients and subjects with normal lungs were studied using the technique of light scattering. The effects of different NaCl concentrations and 6 M guanidine hydrochloride on the molecular size of mucins, their ability to form aggregates, and their shape were investigated. Under the concentration range studied (0.05-3.5 mg/ml), in buffered 0.03 and 0.01 M NaCl, the CF mucins had higher molecular weights (12.2 x 10(6) to 17.1 x 10(6) and 9.5 x 10(6) to 10.4 x 10(6), respectively) than those observed in buffered 0.15 M NaCl (4.3 x 10(6) to 6.6 x 10(6]. These results were interpreted in terms of CF mucins self-aggregating in buffered 0.03 and 0.01 M NaCl. In contrast, in the both buffered 0.3 and 0.15 M NaCl, the normal respiratory mucins had molecular weights of 6.3 x 10(6) to 8.6 x 10(6), thus suggesting the absence of normal mucin aggregation in buffered 0.03 M NaCl. In the presence of 6 M guanidine HCl both CF and normal mucins had molecular weights of about 5 x 10(6) and showed more extended structure (i.e., larger radius of gyration) than in the presence of 0.03 or 0.15 M NaCl. Studies of the relationship of the light scattering intensity with scattering angle showed that, under the above experimental conditions studied, both CF and normal respiratory mucins were polydisperse flexible coil-shaped molecules. The increased aggregation of CF mucins observed at lower salt concentrations may alter the viscoelastic properties of CF lung mucus secretions.

Role of directional Ca 2+ effect on reduced viscosities of mucus secretions from chicken trachea in vitro

Reduced viscosities of fibrillar and gelatinous type mucins produced in response to high submucosal Ca 2+ and low luminal Ca 2+ effects were significantly higher than those of corresponding types of normal mucins. The increased reduced viscosity of experimental mucin samples was due to their aggregation with unique low molecular weight ( M r 325 000 and 46 200) sulphate-rich component. The Ca 2+ appeared to exert two opposing effects on viscosity properties of mucins; whereas Ca 2+ through simple ionic interactions decreased the reduced viscosity of mucins, its ability to promote macromolecular mucin complex formation increased their reduced viscosity. Formation of Ca 2+-dependent complexes between different types of mucins appeared to be a selective phenomenon between sulphate-rich mucins and components. Ester sulphate residue content rather than N-acetylneuraminic acid residue content of these mucins and low molecular weight components showed a very good correlation with their reduced viscosity and Ca 2+-binding values.