The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.     

Porcine submaxillary mucin contains a cystine-rich, carboxyl-terminal domain in addition to a highly repetitive, glycosylated domain

The sequence of a 3.65-kilobase cDNA encoding a large portion of the polypeptide chain of porcine submaxillary mucin (apomucin) has been completed. The encoded polypeptide contains 1150 residues with the carboxyl-terminal 240 residues forming a globular domain that is rich in half-cystine, but deficient in sites for oligosaccharide attachment. The remaining 910 residues preceding the half-cystine-rich domain appear devoid of secondary structures, but they are rich in serine and threonine to which the O-linked oligosaccharides are bound. The first 391 residues of apomucin contain several tandemly repeated, identical sequences of 81 residues. Blots of genomic DNA partially digested with restriction nucleases show that at least 25 of these identical repeats are present in apomucin. The amino acid composition of apomucin isolated in the absence of protease inhibitors was shown earlier (Eckhardt, A. E., Timpte, C. S., Abernethy, J. L., Toumadje, A., Johnson, W. C., Jr., and Hill, R. L. (1987) J. Biol. Chem. 282, 11339-11344) to be devoid of half-cystine. In contrast, the amino acid composition of mucin purified in the presence of protease inhibitors contains half-cystine in amounts predicted by the cDNA sequence and also suggests that this mucin has about 25 tandem repeats. Thus, apomucin contains at least 2800 amino acid residues. Moreover, immunoblots of apomucin prepared in the presence or the absence of protease inhibitors, with antibodies specific for the half-cystine-rich domains or the tandem repeat sequences, show that the half-cystine-rich domain is absent in apomucin unless protease inhibitors are present throughout. Both types of mucin, however, contain the highly repetitive sequences. The molecular weight of undegraded apomucin has not been established exactly, but gel filtration in 6 M guanidine hydrochloride suggests that it is considerably higher than 250,000. RNA blot analysis shows that apomucin mRNA is large and polydisperse in accord with the message size necessary to synthesize the large apomucin polypeptide. These structural features of apomucin suggest a model for the structure of the mucin molecule that correlates well with its reported properties.     

Influence of the amino acid sequence on the MUC5AC motif peptide O-Glycosylation by Human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s)

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamine to human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.     

Structural properties of porcine submaxillary gland apomucin

Porcine submaxillary gland mucin was deglycosylated with a mixture of pure glycosidases to give apomucin containing less than 1% carbohydrate. The resulting apomucin freed of glycosidases was found to contain nine amino acids: threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, and arginine. Serine, threonine, glycine, and alanine comprise 77% of the composition. The molecular weight of apomucin was 96,500 as determined by gel filtration in guanidine hydrochloride. Its Stokes radius was greater than 68.6 A, a far larger value than expected for a globular protein with Mr = 96,500. Circular dichroism spectroscopy of apomucin suggests that it contains 42% aperiodic or "other" structure, 40% beta-turns, 10% antiparallel pleated sheet, and 8% helical structures. The predicted secondary structure of a 50-residue peptide from ovine submaxillary gland mucin resembles the circular dichroism predictions, being dominated by turns that would lead to an extended nonglobular structure. Analysis for the secondary structure of a 36-residue tryptic peptide derived from porcine submaxillary gland apomucin predicts a similar structure. It is concluded that apomucin is likely devoid of traditional secondary structure and serves as a scaffold upon which oligosaccharides are added in O-glycosidic linkage. When sufficient sialic acid is present in the oligosaccharides, native highly viscous mucin containing about two-thirds carbohydrate by weight is obtained.     

Synthesis and macromolecular organization of gastrointestinal mucin.

Mucus glycoproteins (mucins), the principal determinants of mucus protective qualities and mucosal defense, are studied extensively to define pathological aberrations in the relation to gastrointestinal disease and to develop the mucous barrier strengthening agents. Recent work from our laboratory provided evidence as to the initial stages of the gastrointestinal mucin synthesis, molecular size of the apomucin, its macromolecular organization and interaction with other elements of gastrointestinal mucus. Using monoclonal antibodies against apomucin (clone 1H7), O-glycosylated with N-acetylgalactosamine apomucin (clone 2B4), and that against carboxyl terminal of the apomucin (clone 3G12), the mucin synthesizing polysomes were isolated and glycosylated peptides ranging in size from 6-60 kDa identified. The in vitro synthesis in the cell-free system also afforded 60-64 kDa products recognized by 1H7 and 3G12 antimucin MAbs. The obtained results provided evidence that the mucin core consists of 60 kDa peptide which at cotranslational stage is O-glycosylated with N-acetylgalactosamine. Studies on mucin polymer assembly revealed that mucin preparations prepared by equilibrium density gradient centrifugation and Sepharose 2B chromatography (Mantle, M., Mantle, D., and Allen, A. (1981) Biochem. J. 195, 277-285) are not completely purified and contain DNA and extraneous proteins. The evidence was obtained that so called mucin "link protein", 118 kDa glycopeptide, is a N-glycosylated fragment of fibronectin, whereas the supposedly native undegraded mucin isolated by Carlstedt et al. (Biochem. J. (1983) 211, 13-22) was found to contain mucin-fibronectin-DNA complexes. The general picture that emerged from the studies is that the pure mucin consists of 60 kDa glycosylated peptides only. The carboxyl terminal (8-12 kDa fragment) of these peptides is not glycosylated (naked) and is responsible for mucin interaction with fibronectin and other fibronectin-like extracellular matrix proteins. While the formation of the mucosal coat depends on many other factors and extracellular components, our findings on mucin structure and interaction with the extracellular matrix proteins provide explanation as to the possible mechanism of mucin adherence to the epithelial surfaces.     

NMR analysis of human salivary mucin (MUC7) derived O-linked model glycopeptides: comparison of structural features and carbohydrate-peptide interactions.

Two series of glycopeptides with mono- and disaccharides, [GalNAc and Galbeta (1-3)GalNAc] O-linked to serine and threonine at one, two or three contiguous sites were synthesized and characterized by 1H NMR. The conformational effects governed by O-glycosylation were studied and compared with the corresponding non-glycosylated counterparts using NMR, CD and molecular modelling. These model peptides encompassing the aa sequence, PAPPSSSAPPE (series I) and APPETTAAPPT (series II) were essentially derived from a 23-aa tandem repeat sequence of low molecular weight human salivary mucin (MUC7). NOEs, chemical shift perturbations and temperature coefficients of amide protons in aqueous and nonaqueous media suggest that carbohydrate moiety in threonine glycosylated peptides (series II) is in close proximity to the peptide backbone. An intramolecular hydrogen bonding between the amide proton of GalNAc or Galbeta (1-3)GalNAc and the carbonyl oxygen of the O-linked threonine residue is found to be the key structure stabilizing element. The carbohydrates in serine glycosylated peptides (series I), on the other hand, lack such intramolecular hydrogen bonding and assume a more apical position, thus allowing more rotational freedom around the O-glycosidic bond. The effect of O-glycosylation on peptide backbone is clearly reflected from the observed overall differences in sequential NOEs and CD band intensities among the various glycosylated and non-glycosylated analogues. Delineation of solution structure of these (glyco)peptides by NMR and CD revealed largely a poly L-proline type II and/or random coil conformation for the peptide core. Typical peptide fragments of tandem repeat sequence of mucin (MUC7) showing profound glycosylation effects and distinct differences between serine and threonine glycosylation as observed in the present investigation could serve as template for further studies to understand the multifunctional role played by mucin glycoproteins.     

O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein

Ceramidase is a key enzyme involved in regulating cellular levels of ceramide, sphingosine, and possibly sphigosine 1-phosphate and thus could modulate sphingolipid signaling. Here we report that O-glycosylation of the mucin-like domain of neutral ceramidases was required for localization to the surface of plasma membranes. The deduced amino acid sequences of the mammalian enzymes contain a serine-threonine-rich domain (mucin box), which follows the signal/anchor sequence, whereas those of bacterial and invertebrate enzymes completely lack a mucin box, suggesting that the specific domain has been acquired during evolution. In HEK293 cells overexpressing ceramidase, the enzyme was not only secreted into the medium after cleavage of the NH(2)-terminal signal/anchor sequence but also localized at the plasma membrane as a type II integral membrane protein. Lectin blot analysis using peanut agglutinin revealed that the mucin box of the enzyme is highly glycosylated with O-glycans. Interestingly, a mutant lacking the mucin box or possible O-glycosylation sites in the mucin box was secreted into the medium but not localized at the surface of the cells. Furthermore, a mucin box-fused chimera green fluorescent protein (GFP), but not GFP itself, with the signal/anchor sequence was distributed on the surface of the cells. These results suggest that O-glycosylation of the mucin box retains proteins on the plasma membranes. We also found that the 112-kDa membrane-bound enzyme from mouse kidney is O-glycosylated, whereas the 94-kDa soluble enzyme from liver is not. These results clearly indicate that post-translational modification of the enzyme with O-glycans is tissue-specific and helps the enzyme to localize at the surface of plasma membranes as a type II membrane protein.     

Oligosaccharide structures of isolated human colonic mucin species

Purified human colonic mucin contains six distinct components which may be separated by DEAE-cellulose chromatography. Past studies defined the structure of oligosaccharide side chains from the most abundant species III, IV, and V which elute at intermediate salt concentrations. In these studies the structures of oligosaccharide side chains liberated from the remaining early and late eluting species I, II, and VI were determined after isolation by sequential conventional and high performance liquid chromatography through combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Mucin species I, II, and VI contained a less varied array of discrete oligosaccharide structures than that observed in the major mucin components. Mucin species I and II contained five and 10 structures, respectively, which account for 68 and 71% of total oligosaccharide content in these fractions. The predominant oligosaccharides of mucin species I included three neutral structures: a disaccharide GlcNAc beta (1-3)GalNAc-ol, a trisaccharide Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol, and a tetrasaccharide GlcNAc beta (1-4)Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol as well as two acidic components representing the sialylated forms of two of these oligosaccharides. Mucin species II contained these same oligosaccharides as well as four additional acidic structures, notably a disaccharide Neu alpha (2-6)GalNAc-ol and a hexasaccharide Gal beta (1-4)GlcNAc beta (1-3)Gal beta (1-4)GlcNAc beta (1-3) (NeuAc alpha (2-6))-GalNAc-ol, not identified in any other mucin species. The late eluting mucin species VI contained at least five discrete neutral oligosaccharides and six major acidic structures. While the majority of these structures had been previously isolated from the earlier eluting mucin species IV and V, species VI also contained di- and trisialylated oligosaccharides not identified in other mucin species. In conjunction with earlier studies of the major mucin species III, IV, and V, these data define the range of oligosaccharide structures present in human colonic mucin. These studies demonstrate that human colonic mucin possesses species with characteristic and distinguishable combinations of oligosaccharides which reflect variations of common core structures.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

Molecular cloning and characterization of the gene encoding rat submandibular gland apomucin,Mucsmg

Mucin glycoproteins are a major constituent of salivary secretions and play a primary role in the protection of the oral cavity. Rat submandibular glands (RSMG) synthesize and secrete a low molecular weight (114 kDa) mucin glycoprotein. We have isolated, partially sequenced, and characterized the gene which encodes the RSMG apomucin. The gene is encoded by three exons of 106 nt, 69 nt, and 991 nt, separated by introns of 921 nt and 12.5 kb. CAAT and TATA elements are present, at –68 and –26, respectively, in the 5 flanking sequence of the RSMG apomucin gene. The tandem repeat domain present in exon III consists of ten tandem repeats of 39 nt encoding the consensus sequence PTTDSTTPAPTTK. Sequence comparison and organization of the nucleic acid sequence encoding the tandem repeats of two alleles for this gene suggests that the apomucin gene has undergone recombinational events during its evolution. No significant sequence similarity was found with other mucin genes, or with other known salivary gland-specific genes. The gene was localized to rat chromosome 14 using somatic cell hybrids that segregate rat chromosomes. Since this, to our knowledge, represents the first RSMG mucin gene cloned, we have designated this geneMucsmg.Abbreviations RSMG rat submandibular gland - RSM rat salivary mucin - GRP glutamine-glutamic-acid rich protein - nt nucleotide - kb kilobase Sequences reported herein have been assigned GenBank accession numbers U33441 and U33442.     

In vivo glycosylation of MUC1 in airway epithelial cells

The O-glycans that decorate mucin glycoproteins contribute to the biophysical and biochemical properties of these molecules and hence their function as a barrier and lubricant on epithelial surfaces. Alterations in mucin O-glycosylation in certain diseases may contribute to pathology. It is known that both the host cell type and the amino acid sequence of the mucin tandem repeat contribute to the O-glycosylation of a mucin molecule. We expressed an epitope-tagged MUC1 mucin cDNA construct in the airway cell line 16HBE14o- and the colon carcinoma cell line Caco2 and used Fast Atom Bombardment Mass Spectrometry to evaluate the contribution of the host cell to differences in O-glycosylation of a single mucin. Many of the glycans detected on the MUC1 mucin were common to both cell types, as would be predicted from biosynthetic constraints. However, MUC1 synthesized in the airway cell line showed comparatively low levels of sialylation but carried a range of oligo-N-acetyllactosamine structures that were not seen in the colon carcinoma cell line.     

Covalent oligomerization of rat gastric mucin occurs in the rough endoplasmic reticulum, is N-glycosylation-dependent, and precedes initial O-glycosylation

Rat gastric mucin undergoes extensive modifications during biosynthesis, including oligomerization, N- and O-glycosylation, and sulfation. We characterized the events in the rough endoplasmic reticulum (RER) and Golgi complex and studied how these steps are interrelated, using specific inhibitors of cellular processes. The mucin precursors oligomerize in the RER by forming intermolecular disulfide bonds. The oligomers comprise a mixture of predominantly di- and trimers of molar ratio 3:2. The oligomerized precursors are transported to the Golgi complex to form mature, oligomeric mucin by extensive O-glycosylation, and sulfation. N-Glycosylation of the precursor is required for efficient oligomerization. Brefeldin A, which inhibits protein transport between RER and Golgi complex, allows oligomerization and concomitantly induces initial O-glycosylation. Oligomerization and egrees from the RER precedes initial O-glycosylation and are therefore independent of the latter process.     

The role of disulphide bonds in human intestinal mucin

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.     

Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes.

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.     

Protein kinase-A-mediated secretion of mucin from human colonic epithelial cells

Both Ca(2+)- and cAMP-mediated second messenger cascades acutely regulate mucin secretion from human colonic epithelial cells. To better understand the cAMP-dependent regulation of mucin secretion we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms in mucus-secreting T84 cells, and determined which of these isoforms is responsible for agonist-stimulated mucin secretion. Our results show the presence of both type I and type II PKA in cells that also contain large mucin granules. Forskolin caused a rapid and sustained increase in PKA activity that reached a maximum 5-10 min following its addition. Secretion of mucin was detected 15 min following exposure to forskolin, and continued to increase for a further 15 min before reaching a plateau. Mucin secretion was also measured in the presence of combinations of site-selective cAMP analog pairs, which preferentially activate either type I or type II PKA. Similar levels of mucin secretion were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase mucin secretion. Thus, activation of either type I or type II PKA is able to maximally stimulate secretion of mucins from T84 human colonic epithelial cells.     

Improvement in separation of system I and system II particles of photosynthesis obtained by digitonin treatment

By a combined use of digitonin treatment and subsequent centrifugation on a linear sucrose density gradient, the whole green material of the chloroplast lamellae was separated into System I and System II particle fractions, leaving no other fractions of intermediate properties at the final step of separation.

Each of these particle fractions obtained had properties characteristic of System I or System II with respect to the molar ratio of chlorophyll a/chlorophyll b, the content of P700, the fluorescence emission spectrum at −196°;, photoreduction activities with ferricyanide and NADP⁺, and induction of fluorescence.

About 40 and 50% of the total chlorophyll in the original chloroplasts were recovered in System I and System II particles, respectively. Only small amounts of total chlorophyll (less than 10%) were found as free chlorophyll detached from the lamellae through the digitonin treatment.

These results support the view that the lamellae of chloroplasts are composed of about equal amounts of System I and System II particles on a chlorophyll basis.     

A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.     

A MUC1 tandem repeat reporter protein produced in CHO-K1 cells has sialylated core 1 O-glycans and becomes more densely glycosylated if coexpressed with polypeptide-GalNAc-T4 transferase

A recombinant mucin O-glycosylation reporter protein, containing 1.7 tandem repeats (TRs) from the transmembrane mucin MUC1, was constructed. The reporter protein, MUC1(1.7TR)-IgG2a, was produced in CHO-K1 cells to study the glycosylation of the MUC1 TR and the in vivo role of polypeptide-GalNAc-T4 glycosyltransferase. N-terminal sequencing of MUC1(1.7TR)-IgG2a showed that all five potential O-glycosylation sites within the TR were used, with an average density of 4.5 glycans per repeat. The least occupied site was Thr in the PDTR motif, where 75% of the molecules were glycosylated, compared to 88-97% at the other sites. This glycan density was confirmed by an alternative liquid chromatography-mass spectrometry (LC-MS) based approach. The O-linked oligosaccharides were released from MUC1(1.7TR)-IgG2a and analyzed by nano-LC-MS and LC-MS/MS. Four oligosaccharides were present, NeuAcalpha2-3Galbeta1-3GalNAcol, NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAcol, Galbeta1-3(NeuAcalpha2-6)GalNAcol, and Galbeta1-3GalNAcol, the two first being most abundant. Coexpression of the human polypeptide-GalNAc-T4 transferase with MUC1(1.7TR)-IgG2a increased the glycan occupancy at Thr in PDTR, Ser in VTSA, and Ser in GSTA, supporting the function of GalNAc-T4 proposed from previous in vitro studies. The expression of GalNAc-T4 with a mutation in the first lectin domain (alpha) had no glycosylation effect on PDTR and GSTA but surprisingly gave a dominant negative effect with a decreased glycosylation to around 50% at the Ser in VTSA. The results show that introduction of glycosyltransferases can specifically alter the sites for O-glycosylation in vivo.     

NetOglyc: Prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility

The specificities of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases which link the carbohydrate GalNAc to the side-chain of certain serine and threonine residues in mucin type glycoproteins, are presently unknown. The specificity seems to be modulated by sequence context, secondary structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. Charged residues were disfavoured at position – 1 and +3. A jury of artificial neural networks was trained to recognize the sequence context and surface accessibility of 299 known and verified mucin type O-glycosylation sites extracted from O-GLYCBASE. The cross-validated NetOglyc network system correctly found 83% of the glycosylated and 90% of the non-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predictions of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based on the amino acid sequence. The server addresses are http://www.cbs.dtu.dk/services/NetOGlyc/ and netOglyc@cbs.dtu.dk.