Database analysis of O-glycosylation sites in proteins

Statistical analysis was carried out to study the sequential aspects of amino acids around the O-glycosylated Ser/Thr. 992 sequences containing O-glycosylated Ser/Thr were selected from the O-GLYCBASE database of O-glycosylated proteins. The frequency of occurrence of amino acid residues around the glycosylated Ser/Thr revealed that there is an increased number of proline residues around the O-glycosylation sites in comparison with the nonglycosylated serine and threonine residues. The deviation parameter calculated as a measure of preferential and nonpreferential occurrence of amino acid residues around the glycosylation site shows that Pro has the maximum preference around the O-glycosylation site. Pro at +3 and/or -1 positions strongly favors glycosylation irrespective of single and multiple glycosylation sites. In addition, serine and threonine are preferred around the multiple glycosylation sites due to the effect of clusters of closely spaced glycosylated Ser/Thr. The preference of amino acids around the sites of mucin-type glycosylation is found likely to be similar to that of the O-glycosylation sites when taken together, but the acidic amino acids are more preferred around Ser/Thr in mucin-type glycosylation when compared totally. Aromatic amino acids hinder O-glycosylation in contrast to N-glycosylation. Cysteine and amino acids with bulky side chains inhibit O-glycosylation. The preference of certain potential sequence motifs of glycosylation has been discussed.     

Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide

The similarity or identity of O-glycosylation in glycoproteins from natural sources or produced in heterologous cell lines, a central problem for the development of many biotechnologically relevant production processes, was examined using interleukin-2 (IL-2) as a model. Human interleukin-2 was constitutively expressed in several mammalian cell lines in high amounts. The recombinant proteins were purified to homogeneity and their carbohydrate structures were analyzed. Only the NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc oligosaccharide structure or the NeuAc alpha 2-3Gal beta 1-3GalNAc were found in all IL-2 preparations secreted from recombinant Ltk-, Chinese hamster ovary, and baby hamster kidney cell lines. The O-linked chains were exclusively linked to Thr in position 3 of the polypeptide chain which is the carbohydrate attachment site in natural human IL-2. The proportions of O-glycosylated versus nonglycosylated forms of the protein secreted by each recombinant cell line were independent of productivity or of cell culture conditions. Our results show that O-glycosylated human IL-2 can be produced by applying recombinant DNA technology in heterologous cell lines with the same type of post-translational modification that is observed for the protein secreted from natural T lymphocytes.     

Specific in vitro O-glycosylation of human granulocyte-macrophage colony-stimulating-factor-derived peptides by O-glycosyltransferases of yeast and rat liver cells.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is O-glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS-1 cells [Ernst, J.F., Mermod, J.-J. and Richman, L.I. (1992) Eur. J. Biochem. 203, 663-667]. Two types of octapeptides encompassing residues 4-11 (peptide 4-11) and variants thereof, or residues 8-15 (peptide 8-15) of hGM-CSF were tested as substrates for in vitro O-glycosylation using dolichyl-phosphate- D-mannose: protein O-D-mannosyltransferase (Man-transferase) of the yeast Saccharomyces cerevisiae, or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) of rat liver cells. Peptide 8-15 was found to be O-glycosylated at residues Ser9 and Thr10 by GalNAc-transferase and, with reduced efficiency, also by Man-transferase. Peptide 4-11 was a good substrate for yeast Man-transferase, leading to mannosylation of only Thr10, whereas it was very poorly O-glycosylated at positions Ser5 and Ser7 by GalNAc-transferase. The observed differences in peptide-acceptor activities indicate that the site of O-glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.     

Effect of carbohydrate structure on biological activity of artificially N-glycosylated eel calcitonin

To reveal the function of the carbohydrate portion of glycopeptides and glycoproteins, we chemo-enzymatically synthesized artificially N-glycosylated derivatives of eel calcitonin and studied their three-dimensional structure and biological activity. The CD and NMR spectra in trifluoroethanol-H₂O solution showed that the glycosylation did not change the three-dimensional structure. All the derivatives retained the strong in vivo hypocalcemic activity of calcitonin. However, the relative activity was dependent on the structure of the attached carbohydrate. The single GlcNAc attachment best enhanced the activity, while larger carbohydrates decreased the activity. This relative activity order of compounds could be partly explained by their calcitonin-receptor binding affinity, though the affinity of the GlcNAc derivative did not exceed that of calcitonin. The enhanced hypocalcemic activity of the GlcNAc derivative was explained by its altered biodistribution. The GlcNAc attachment caused calcitonin to escape from the trap at the liver during the early circulation. Thus, the glycosylation was shown to modulate the biological activity of calcitonin depending on the carbohydrate structure without a change in the peptide backbone conformation.     

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.     

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The degradation of glycoproteins with lithium borohydride. Isolation and analysis of O-glycopeptides with reduced C-terminal amino acid residue

By the example of fetuin and a blood-group-specific mucin from porcine stomach, we showed that, under conditions of reductive degradation of glycoproteins with LiBH4-LiOH in 70% aqueous tert-butyl alcohol, the reduction and cleavage of amide bonds occur much faster than the simultaneous beta-elimination of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are the O-glycosylated derivatives of 2-aminopropane-1,3-diol and 2-aminobutane-1,3-diol (the products of reduction of glycosylated Ser and Thr) and the glycopeptides containing 2-4 amino acid residues with reduced C-terminal amino acid. Seventeen homogeneous O-glycopeptides were isolated from the fetuin degradation products by ion-exchange and reversed-phase HPLC. Their structures were determined by MALDI-TOF mass spectrometry and by analyses for amino acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation sites in O- and N,O-glycoproteins is discussed.     

An NMR study of O-glycosylation induced structural changes in the alpha-helix of calcitonin

We previously reported that two out of seven artificially O-glycosylated calcitonin derivatives had an altered peptide backbone conformation as indicated by decreased helical contents, determined by CD measurement. In the present study, two of those derivatives, in which a GalNAc residue is attached to Thr6 or Thr21 of calcitonin, were analyzed by NMR in order to determine the structural changes induced by the O-glycosylation in more detail. Deviations in the chemical shifts suggest that the structural change is not global but only a local one and is located in the vicinity of each O-glycosylation site. The intensities of the NOE cross peaks, an indicator of -helical structure, also were decreased around the O-glycosylation site. The hydrogen/deuterium exchange rates of the main chain amide protons increased at the N- or C-terminal portion of the -helix corresponding to the respective O-glycosylation site and explains the results of the CD experiments. The inter-residual NOE cross peaks between the carbohydrate and the peptide portions, other than the O-glycosylated amino acid residue, showed that local structural contacts extended three or two residue distance for Thr6- or Thr21-glycosylated derivative, respectively. Thus, we conclude that the O-glycosylation induced a change in the local structure and that this structural perturbation modulated the original -helical structure of calcitonin, resulting in the apparent decrease in the helical content deduced from CD spectra.     

Biosynthesis of truncated O-glycans in the T cell line Jurkat. Localization of O-glycan initiation

Glycoproteins from the human T leukemia cells Jurkat were found to bind to the GalNAc alpha 1----Ser/Thr-specific lectin from Salvia sclarea seeds. The analysis of the O-linked saccharides of immunopurified leukosialin, the major [3H]glucosamine-labeled glycoprotein in Jurkat cell lysate, revealed the presence of mainly GalNAc alpha 1----Ser/Thr with only minor amounts (approximately 17%) of more complex O-glycans. A comparison between Jurkat and K562 cell glycosyltransferase involved in the biosynthesis of O-linked carbohydrates showed that a markedly lower activity of UDP-Gal:GalNAc alpha 1----Ser/Thr beta 1----3galactosyltransferase is apparently responsible for the presence of truncated O-glycans in the Jurkat cell line. The O-glycosylation defect makes Jurkat cells an ideal model to study the initiation of O-linked saccharides. Pulse-chase experiments with [35S] methionine showed that the addition of GalNAc to leukosialin is responsible for the decreased mobility of the mature glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, no biosynthetic intermediates between the O-glycan-free precursor and the fully O-glycosylated form could be detected either with an anti-leukosialin antiserum or with the GalNAc-specific lectin. Lowering the chase temperature to 15 degrees C completely inhibited the transfer of GalNAc to the peptide core indicating that O-glycan initiation takes place in the first Golgi elements and not in transitional vesicles between endoplasmic reticulum and Golgi. In addition, treatment of the cells with monensin did not inhibit GalNAc transfer to leukosialin apoprotein. These results indicate that the initiation of O-glycosylation in Jurkat cells starts in the cis-Golgi stacks.     

Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.     

The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities

The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Differences in kinetic properties, substrate specificities, and expression patterns of these isoenzymes provide for differential regulation of O-glycan attachment sites and density. Recently, it has emerged that some GalNAc-transferase isoforms in vitro selectively function with partially GalNAc O-glycosylated acceptor peptides rather than with the corresponding unglycosylated peptides. O-Glycan attachment to selected sites, most notably two sites in the MUC1 tandem repeat, is entirely dependent on the glycosylation-dependent function of GalNAc-T4. Here we present data that a putative lectin domain found in the C terminus of GalNAc-T4 functions as a GalNAc lectin and confers its glycopeptide specificity. A single amino acid substitution in the lectin domain of a secreted form of GalNAc-T4 selectively blocked GalNAc-glycopeptide activity, while the general activity to peptides exerted by this enzyme was unaffected. Furthermore, the GalNAc-glycopeptide activity of wild-type secreted GalNAc-T4 was selectively inhibited by free GalNAc, while the activity with peptides was unaffected.     

Naturally occurring structural isomers in serum IgA1 o-glycosylation

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of O-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr(225), Thr(228), Ser(230), Ser(232) and Thr(236) have been identified as the predominant sites of O-glycan attachment. The range and distribution of O-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 O-glycan heterogeneity by use of high resolution LC-MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 O-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the O-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable O-glycan chains attached to Ser(230), Thr(233) or Thr(236) produce the predominant positional isomers, including O-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a single sample, and have implications for defining epitopes based on clustered O-glycan variability.     

Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments

In-gel digestion of densely O-glycosylated proteins, an essential step in proteome analysis, is often hampered by steric hindrance of the proteases. To overcome this technical problem a simple and convenient method has been developed, which combines several advantages: (1) Approximately 70% of the oligosaccharides are cleaved without significant protein hydrolysis at the optimal reaction conditions of 70% ethylamine, and quantitative cleavage is achieved with 40% methylamine, at 50 degrees C. (2) To the unsaturated derivatives of Ser and Thr the alkylamine is added as a label of previous O-glycosylation sites. (3) The alkylaminylated protein is effectively cleaved by proteolysis. (4) The modified peptides are identified by MALDI mass spectrometry under consideration of incremental mass increases. (5) The alkylamine label is stable under MALDI post-source-decay analysis as well as in collision-induced dissociation experiments allowing sequencing and peptide localization of O-glycosylation sites. Applicability of the method is evaluated with a series of synthetic glycopeptides, the densely O-glycosylated human glycophorin A, and with the mucin MUC1 from human milk fat globule membranes.     

Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides

We present evidence that site-specific O-glycosylation by recombinant polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 is controlled by the primary sequence context, as well as by the position and structure of previously introduced O-glycans. Synthetic mucin-type (glyco)peptides corresponding to sections of the tandem repeat regions of MUC1, MUC2, and MUC4 were used as substrates for recombinant polypeptide N-acetylgalactosaminyltransferases, rGalNAc-T2 and -T4. By concerted and sequential action the two transferases are able to fully glycosylate MUC1 but only partially MUC2 and MUC4 tandem repeat peptides. GalNAc residues on MUC1 acceptor peptides trigger activity of rGalNAc-T4 directed to Ser in VTSA and Thr in PDTR and of rGalNAc-T2 to Ser/Thr within the GSTA motif of variant MUC1 peptides. However, elongation of GalNAc by beta3-galactosylation inhibits rGalNAc-T4 activity completely and rGalNAc-T2 activity with respect to the acceptor site GSTA. These findings are in accord with the inhibition of rGalNAc-T2 and -T4 by fully GalNAc-substituted MUC1 repeat peptide and support a glycosylation-dependent activity induction or enhancement of both enzymes.     

O-糖基化起始糖基转移酶的活性与结构研究

多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T） 是催化N-乙酰氨基半乳糖（GalNAc）结合到蛋白质Ser或Thr上的糖基转移酶,是黏蛋白型O-糖基化修饰的起始糖基转移酶。ppGalNAc-T是一个酶家族,表达产物均为Ⅱ型膜蛋白。虽然氨基酸序列高度同源,但各成员具有独特的底物特异性和动力学特征。因此,ppGalNAc-T的底物作用机制是O-糖基化研究领域中的关键课题。近年来,通过利用定点突变及晶体结构解析技术,ppGalNAc-T中与底物相互作用的重要氨基酸残基以及由这些残基所形成的对底物结合起关键作用的空间构象逐渐被揭示,为了解ppGalNAc-T酶家族的底物作用机制及其蛋白结构与催化活性间的关系提供了理论依据。     

Concepts and Principles of O-Linked Glycosylation

The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucin-type O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosac-charides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.     

Specificity of Amaranthus leucocarpus syn. hypocondriacus lectin for O-glycopeptides

Amaranthus leucocarpus syn. hypochondriacus lectin (ALL) has been shown to be specific for N-acetyl-D-galactosamine (GalNAc). In this work, we determined a value of 1.0 x 10(-2) M for the association constant of ALL for GalNAc, calculated using fluorescence spectroscopy assays. Using neoglycopeptides obtained by in vitro O-glycosylation, we determined the main features of O-glycopeptides recognized by ALL using molecular dynamics simulations, capillary electrophoresis, and ELISA. Neo-glycopeptides were obtained by in vitro O-glycosylation reaction using microsomal preparations of murine thymocytes, human gastric fundus and colonic mucosa. ELISA assays were performed with peroxidase-labeled murine monoclonal IgG2, kappa light chain (5D4) antibodies against ALL. Among the in vitro neoglycopeptides, only those of TTSAPTTS containing GalNAc at Thr in #2 and #6 reacted with ALL. Neither the TTSAPTTS glycopeptide, containing a unique GalNAc residue at Thr in #2, nor others (with more than two GalNAc residues) interacted with the lectin. Computational docking assays of the lower energy conformers for interactions between glycopeptides and lectins confirmed that ALL recognized GalNAc residues when they are spaced out in glycan structures, whereas GalNAc residues arranged in clusters prevented interaction with the lectin, indicating that ALL is specific for a special GalNAc-containing motif found in different O-glycoproteins.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Conformational flexibility and biological activity of salmon calcitonin

We have assessed the biological activity of salmon calcitonin I (sCT) using an in vivo biological assay of hypocalcemic activity in rats. The changes in biological activity observed are explained on the basis of changes in the conformational properties of the hormone analogues. Helical content in the presence and absence of lipids and detergents was assessed by using circular dichroism, and the section of the molecule that folds into a helix was predicted on the basis of the helix-coil transition theory of Mattice and co-workers. In the amino acid sequence of sCT, residue 8 is valine and residue 16 is leucine. The synthetic calcitonin derivatives [Gly8]sCT and [Ala16]sCT have higher biological activity than the native hormone although they have a lower helical content. The increased biological activity of these derivatives is ascribed to an increase in their conformational flexibility resulting from the substitution of amino acid residues with less bulky side chains and less tendency to form helical structures. The derivative [Met8]sCT has less substitution than sCT on the beta-carbon at position 8, but it has increased helix-forming potential in the region of residues 8-12. These two factors affect conformational flexibility in opposite ways, resulting in the biological activity of [Met8]sCT being slightly higher than that of sCT. However, increased conformational flexibility does not always increase biological activity. Substitution of the L-arginine at residue 24 for a D-arginine has little effect on the conformational properties or biological activity of sCT. However, [Gly8, D-Arg24]sCT is less active than sCT, [Gly8]sCT, or [D-Arg24]sCT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom–up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom–up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins.