Structural studies on the glycoproteins from bovine cervical mucus.

The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely 'naked' peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.     

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.     

Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain.

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.     

Growth of enveloped RNA viruses in a line of chinese hamster ovary cells with deficient N-acetylglucosaminyltransferase activity.

Sindbis and vesicular stomatitis viruses were grown in a line (termed 15B) of Chinese hamster ovary (CHO) cells that is deficient in a specific UDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosaminyltransferase. Both viruses replicated normally in the cell line, but the glycoproteins of the released virus migrated faster on sodium didecyl sulfate-polyacrylamide gels than did glycoproteins of virus grown in parent CHO cells. Digestion of the viral glycoproteins with Pronase followed by gel filtration demonstrated that the glycoproteins with Pronase followed by gel filtration demonstrated that the glycopeptides of Sinbis-15B virus were much smaller than the glycopeptides of Sindbis-CHO virus. In addition, Sindbis-15B viral glycopeptides but not Sindbis-CHO viral glycopeptides contained terminal alpha-mannose residues as shown by their susceptibility to alpha-mannosidase digestion. These findings demonstrate that the oligosaccharide units of the glycoproteins of vesicular stomatitis and Sinbis viruses are altered when the viruses are grown in 15B cells. We conclude that the N-acetylglucosaminyltransferase that is missing in 15B cells normally participates in the biosynthesis of the oligosaccharide units of the viral glycoproteins, and in the absence of this enzyme incomplete oligosaccharide chanis are produced. Viruses released from 15B cells appear to retain full infectivity; Sindbis-15B virus, however, showed a significant decrease in hemagglutination titer compared with that of Sindbis-CHO virus.     

Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.     

Proteolytic release of glycopeptides from glycoproteins transferred to nitrocellulose following gel electrophoresis

To determine whether glycopeptides could be released from glycoproteins bound to nitrocellulose, the glycoproteins of murine mammary tumor virus (MuMTV) were radiolabeled by the periodate oxidation/tritiated sodium borohydride reduction technique and separated by gel electrophoresis followed by diffusion transfer. Pronase digestion of nitrocellulose filter strips containing labeled glycoproteins (gp55 or gp34) revealed a rapid release of glycopeptides, i.e., approximately total release within 4 h. The released glycopeptides were similar in size, as determined by molecular sieving chromatography, to glycopeptides obtained by proteolytic digestion of MuMTV glycoproteins from dried gel strips (A. Zilberstein et al., 1980, Cell 21, 417-427) or in solution (M. J. Yagi et al., 1978, Virology 91, 291-304).     

The isolation and characterization of a concanavalin A receptor from boar spermatozoa surface

Boar spermatozoa were radioactively labeled by either lactoperoxidase-catalysed iodination or galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. Plasma membrane glycoproteins were solubilized with the non-ionic detergent Nonidet P40 and separated by affinity chromatography on concanavalin A-Sepharose. A major water-soluble concanavalin A receptor of molecular weight greater than 160 000 was isolated by gel filtration and ion-exchange chromatography. Its amino acid and carbohydrate composition were determined. This glycoprotein is susceptible to digestion by trypsin or chymotrypsin.     

The major sialoglycoprotein of human T-lympocytes

Human, peripheral-blood T-lymphocytes and human, T-lymphoblastoid cells of a MOLT 4B cell-line were surface-labeled by lactoperoxidase-catalyzed iodination, periodate and sodium borotritide, and galactose oxidase and sodium borotritide, and analyzed by dodecyl sodium sulfate-polyacrylamide gel-electrophoresis. Both types of cells were found to show a major, cell-surface sialoglycoprotein with an apparent mol. wt. of 95 000. After neuraminidase treatment, this glycoprotein showed a higher mol. wt. of 120 000. The major sialoglycoprotein of both types of cells bound to wheat-germ agglutinin and concanavalin A and, after neuraminidase treatment, to Arachis hypogaea agglutinin. The glycopeptides obtained from these glycoproteins by Pronase digestion gave similar elution-profiles on Sephadex G-50 gel filtration. These results suggest that the major sialoglycoprotein of normal T cells and that of MOLT 4B cells are very similar, if not identical.     

Role of sialic acid and sulfate groups in cervical mucus physiological functions: study of Macaca radiata glycoproteins

The influence of charged groups in glycoproteins was investigated to assess their effect on the physiological functions of bonnet monkey cervical mucus. The macromolecular glycoproteins from peri-ovulatory, midcycle phase cervical mucus were treated with Pronase, trypsin and chymotrypsin and the enzyme-resistant glycoproteins purified by gel filtration on Sepharose 4B and a high molecular weight component containing carbohydrates, proteins and sulfate groups was recovered in high yield. This material still reacted with an antiserum directed against purified midcycle glycoprotein but not against another antiserum directed against luteal phase purified glycoproteins. Upon treatment with Pronase, trypsin and chymotrypsin, asialoglycoproteins and desulfated asialoglycoproteins released fragments of low molecular sizes, none of which reacted with the anti-midcycle glycoprotein antiserum. Cervical mucus collected from the estrogenic phase displayed a morphology supporting sperm migration, and this mucus retains the same morphology and reacts with the anti-midcycle glycoprotein antiserum following mild treatment with sialidase and subsequently with Pronase. These results imply that charged carbohydrate groups help maintain the structural and functional integrity of the mucus glycoprotein in its biological environment.     

Studies on bonnet monkey cervical mucus the effect of proteases on mucus glycoproteins of macaca radiata

The influence of proteinases on monkey cervical glycoproteins was investigated to assess their effect on cervical mucus and, thereby, on sperm penetration. The major component of periovulatory cervical mucus, a high molecular weight glycoprotein, was treated with Pronase, trypsin, chymotrypsin, papain, and bovine seminal peptidase, and the enzyme-resistant glycoprotein was purified by gel filtration on Sepharose 2B. A macromolecular component in high yield was recovered containing carbohydrate and protein moieties. Asialoglycoprotein, on treatment with Pronase, trypsin, and bovine seminal peptidase released more than one glycoprotein fragment. The carbohydrate and amino acid components of the native and degraded glycoproteins were similar in composition with variations in proportions. The structure of the carbohydrate-rich, pronase-resistant glycoprotein, further purified on Sepharose 2B, was examined. Sequential Smith degradation and methylation of the degraded glycoprotein fragment established a structure that shows some differences to that of the native glycoprotein. The influence of proteinases on cervical-mucus glycoproteins and a possible mechanism of sperm penetration through Pronase-treated glycoproteins is discussed.     

Stimulation of the biosynthesis of membrane glycoproteins from Zajdela ascites hepatoma cells by Robinia lectin.

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis.

Previous studies have shown that human small-intestinal mucin consists of high-Mr glycoproteins and a smaller S-S-bonded protein of 118 kDa. The major antigenic determinants of the mucin were associated with the large glycoproteins, but depended for stability on intact disulphide bonds, and were destroyed by digestion with Pronase. In the present study we isolated and analysed the component parts of mucin from patients with cystic fibrosis with special attention being paid to the peptide constituents. After reduction with 0.2 M-beta-mercaptoethanol [5 min, 100 degrees C in 1% SDS (sodium dodecyl sulphate)], the large glycoproteins and smaller peptide with an apparent molecular size of 118 kDa were separated by equilibrium density-gradient centrifugation in CsCl, Sepharose 4B chromatography or preparative SDS/polyacrylamide-gel electrophoresis. The large glycoproteins contained about 70% of the protein of the native mucin. Digestion with Pronase resulted in a further loss of 'naked' protein (10% of the native mucin protein) from the C-terminal end of the glycoprotein peptide core, and left behind highly glycosylated proteins comprised mainly (70 mol%) of threonine, serine and proline. The 118 kDa component, which contained about 30% of the native mucin protein, consisted mainly of aspartic acid, serine, glutamic acid and glycine (40 mol%), plus threonine, proline, alanine, valine and leucine (35 mol%). Together with the 'naked' protein segment, the 118 kDa component contained most of the cysteine residues of the native mucin. Surprisingly, the peptide also contained carbohydrate (less than or equal to 5% of the native mucin carbohydrate but 50% by weight of the 118 kDa component), which included 9 mol% mannose, suggesting the presence of N-linked oligosaccharides. The peptide exhibited strong non-covalent interactions with the high-Mr glycoproteins and a tendency to self-aggregate in the absence of dissociating agents. Our findings therefore suggest that native mucin consists of large glycoproteins capable of forming disulphide bridges from their C-terminal 'naked' (antigenic) regions to a smaller glycopeptide having an Mr of 118 000.     

Isolation and characterization of a sulfated glycoprotein from rabbit uterus.

Crude glycoproteins were extracted with 0.15 M NaCl from the pooled endometrial scrapings of rabbit uteri after treatment with estrogen. The crude glycoproteins were fractionated with ammonium sulfate, followed by DEAE-cellulose column chromatography, treatment with CM-Sephadex C-25 and gel filtration on Sephadex G-200. Subsequently, purification of an acidic glycoprotein was carried out by gel filtration on Sephadex G-200 and then Sepharose 4B. The results of electrophoresis and enzymatic digestion, together with analytical data and the infrared spectrum indicated that the acidic glycoprotein was a sulfated glycoprotein.     

Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG.2

gG.2 glycoprotein was purified by H966 monoclonal antibodies linked to Sepharose from herpes simplex virus type 2-infected HEp-2 cells labeled with [3H] glucosamine. The glycoprotein was subjected to Pronase digestion and the glycopeptides were fractionated by Con A-Sepharose in a major fraction (88.5% of total radioactivity) unbound to the lectin gel and in a minor species which bound to the lectin as a N-linked diantennary oligosaccharide. Mild and strong acid hydrolysis of Con A-unbound and Con A-bound fractions revealed that (i) both species were highly sialylated; (ii) the Con A-unbound fraction contained mainly labeled N-acetylgalactosamine, as is the case for O-linked oligosaccharides; and (iii) the Con A-bound fraction carried the vast majority of the labeled N-acetylglucosamine present in gG.2. Three size classes of oligosaccharides were separated from mild alkaline borohydride-treated Con A-unbound glycopeptides, which accounted for about 80% of the radioactivity present in the fraction. Galactosaminitol was recovered as the major labeled product in the strong acid hydrolyzates of the oligosaccharides generated by reductive beta-elimination, indicating that they were O-glycosidically linked to the peptide backbone. Thin-layer and DEAE-Sephacel chromatography of the three O-linked oligosaccharide species indicated that disialylated tetrasaccharides and monosialylated trisaccharides were the major components, whereas neutral disaccharide was a minor component. Digestion with neuraminidase and beta-galactosidase of the O-linked oligosaccharides supported the idea that the common disaccharide core was mainly of the structure beta-galactosyl-N-acetylgalactosamine. The large occurrence of O-linked oligosaccharides differentiates this type 2-specific herpes simplex virus glycoprotein from the type-common herpesvirus glycoproteins gB, gC, and gD.     

Differences between the carbohydrate units of cell-surface glycoproteins of moust B- and T-lymphocytes.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.     

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6; characterization of intermediate- and end products

Conglutins, the major peanut allergens, Ara h 2 and Ara h 6, are highly structured proteins stabilized by multiple disulfide bridges and are stable towards heat-denaturation and digestion. We sought a way to reduce their potent allergenicity in view of the development of immunotherapy for peanut allergy. Isoforms of conglutin were purified, reduced with dithiothreitol and subsequently alkylated with iodoacetamide. The effect of this modification was assessed on protein folding and IgE-binding. We found that all disulfide bridges were reduced and alkylated. As a result, the secondary structure lost α-helix and gained some β-structure content, and the tertiary structure stability was reduced. On a functional level, the modification led to a strongly decreased IgE-binding. Using conditions for limited reduction and alkylation, partially reduced and alkylated proteins were found with rearranged disulfide bridges and, in some cases, intermolecular cross-links were found. Peptide mass finger printing was applied to control progress of the modification reaction and to map novel disulfide bonds. There was no preference for the order in which disulfides were reduced, and disulfide rearrangement occurred in a non-specific way. Only minor differences in kinetics of reduction and alkylation were found between the different conglutin isoforms. We conclude that the peanut conglutins Ara h 2 and Ara h 6 can be chemically modified by reduction and alkylation, such that they substantially unfold and that their allergenic potency decreases.     

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

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.     

Polymeric structure of pig small-intestinal mucus glycoprotein. Dissociation by proteolysis or by reduction of disulphide bridges.

Pig small-intestinal mucus glycoprotein, of molecular weight 1.72 X 10(6), is cleaved by Pronase digestion into glycoprotein subunits of molecular weight 4.5 X 10(5). Of the protein component of the native glycoprotein 29% by weight was lost on Pronase digestion, with no loss of carbohydrate. The non-glycosylated region of the protein that was lost with proteolytic digestion had a broad spectrum of amino acid residues, in contrast with the glycosylated region of the protein, which was resistant to proteolysis and was rich in serine, threonine and proline residues. Reduction with 0.2M-mercaptoethanol dissociated the Pronase-digested glycoprotein subunits into smaller glycoprotein subunits of molecular weight 2.7 X 10(5). On reduction, the native glycoprotein was dissociated into subunits of molecular weight 2.4 X 10(5), a similar size to those obtained from reduction of the Pronase-digested glycoprotein. On reductive dissociation of the native glycoprotein, in addition to glycoprotein subunits, protein was also released principally as a component of 90000 molecular weight. This protein was separated quantitatively from the reduced glycoprotein in amounts compatible with one 90000-mol.wt. protein molecule per 1.72 X 10(6)-mol.wt. native glycoprotein molecule. No 90000-mol.wt. protein was released on reduction of the isolated Pronase-digested glycoprotein. Pig small-intestinal mucus glycoprotein is therefore a covalent polymer of glycoprotein subunits joined by disulphide bridges. This polymeric structure differs in important respects from that previously shown for gastric mucus, in particular with respect to the size and number of component subunits per native molecule.