Non-Covalent Forces Hold Glycoprotein Molecules Together in Mucous Gel

Goblet-cell mucin (mucin 1) was isolated and purified from human small-intestinal scrapings. After application of mucin 1 to DEAE-Bio-Gel (A) columns, most of the glycoprotein (76-94% of hexoses) was eluted in the first peak (designated mucin 2). Minor amounts of acidic glycoproteins were eluted with 0.2m- and 0.4m-NaCl in later peaks. Analyses of mucin 1 and mucin 2 revealed mucin 2 to be a monodisperse highly glycosylated glycoprotein containing 6.3% by wt. of protein, N-acetylgalactosamine, N-acetylglucosamine, galactose and fucose. Mucin 1 was similar in composition, but was polydisperse and contained more protein (12.3% by wt.) as well as N-acetylneuraminic acid. Analytical CsCl-gradient ultracentrifugation showed both mucin 1 and mucin 2 to have a major component with an average buoyant density of 1.47000g/ml. Mucin 1 also contained a slightly less-dense minor glycoprotein component. After exhaustive reduction and alkylation mucin 1 retained its major component, but partly dissociated into two lighter glycoprotein components. Mucin 2, in contrast, did not change its density distribution after reduction. Band ultracentrifugation in (2)H(2)O-containing iso-osmotic buffers showed that mucin 1 contained a major fast-sedimenting component (s(o)=37+/-2S), and a minor amount of a slower-sedimenting component. After reduction there was an increased quantity of the latter component, for which an s(o) value of 14.5S was calculated. In contrast, mucin 2 was unaltered by reduction (s(o)=33+/-2S). These findings indicate that the major component of goblet-cell mucin (mucin 2) does not dissociate after S-S-bond reduction, and thus does not apparently rely for its polymeric structure on the association of subunits through covalent disulphide bonds. However, the effects of reduction on mucin 1 suggest that in the native mucin intramolecular disulphide bonds in the minor glycoproteins may stabilize their structure, permitting secondary non-covalent interactions to develop with the major dense mucin (mucin 2) protein.

Section: Discussioncontrasting

confidence: 76%

The role of disulphide bonds in human intestinal mucin

Forstner

Jabbal

Qureshi

et al. 1979

“…This causes the molecule to dissociate into four subunits similar in size to those obtained by reductive cleavage in 2-mercaptoethanol (Snary et al, 1970). Both the viscous and gela--tinous properties (Robson et al, 1975) of the gastric glycoprotein are lost on either proteolytic digestion or reductive cleavage in 2-mercaptoethanol, showing -that the four-subunit polymeric structure ofthe glycoprotein is essential for the rheological properties of both it and the gastric mucus.…”

Section: Results Of Enzyme Digestionmentioning

confidence: 99%

“…The scrapings of pig gastric mucosa were collected and homogenized to solubilize the gel (Robson et al, 1975). The glycoprotein was isolated from the soluble gel by equilibrium-density-gradient centrifugation in CsCl (initial density 1.42g/ml), followed by gel filtration on Sepharose 4B (Starkey et al, 1974).…”

Section: Purification and Characterization Ofpig Gastric Glycoproteinmentioning

confidence: 99%

“…2 x 106) possessing A and H blood-group activity has been shown to be the component that is responsible for the viscous and gelling properties of pig gastric mucus (Allen & Snary, 1972;Allen, 1977). This glycoprotein can be solubilized by homogenization of the mucous gel and isolated free of contaminating protein by equilibrium-density-gradient centrifugation in CsCI (Starkey et al, 1974;Robson et al, 1975). The purified glycoprotein has a protein core, 13% by weight of the glycoprotein, with carbohydrate side chains attached.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The action of proteolytic enzymes on the glycoprotein from pig gastric mucus

Scawen¹,

Allen²

1977

139

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.

“…Potentially destructive methods such as sonication and highspeed homogenization were thus avoided for the solubilization of the mucus gel. High-speed homogenization apparently does not degrade gastric mucins (Robson et al, 1975). In contrast, cartilage proteoglycans are markedly degraded by this treatment (Sajdera & Hascall, 1969).…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of human cervical-mucus glycoproteins

Carlstedt¹,

Lindgren²,

Sheehan³

et al. 1983

223

188

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 X 10(6) and 5.9 X 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).