Structural effects of O-glycosylation on a 15-residue peptide from the mucin (MUC1) core protein

To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, the 15-residue peptide PPAHGVTSAPDTRPA (PPA15) from the MUC1 protein core and its analogue PPA15(T7), glycosylated with alpha-N-acetylgalactosamine on Thr7, were prepared and investigated by NMR spectroscopy. The peptide contains both the GVTSAP sequence, which is an effective substrate for GalNAc-T1 and -T3 transferases, and the PDTRP fragment, which is a well-known immunodominant epitope recognized by several anti-MUC1 monoclonal antibodies. Useful structural results were obtained in water upon decreasing the temperature to 5-10 degrees C. The sugar attachment slightly affected the conformational equilibrium of the peptide backbone near the glycosylated Thr7 residue. The clustering of low-energy conformations for both PPA15 and PPA15(T7) within the GVTSAP and APDTRP fragments revealed structural similarities between glycosylated and nonglycosylated peptides. For the GVTSAP region, minor but distinct clusters formed by either PPA15 or PPA15(T7) conformers showed distinct structural propensities of the peptide backbone specific for either the nonglycosylated or the glycosylated peptide. The peptide backbone of the APDTRP fragment, which is a well-known immunodominant region, resembled an S-shaped bend. A similar structural motif was found in the GVTSAP fragment. The S-shaped structure of the peptide backbone is formed by consecutive inverse gamma-turn conformations partially stabilized by hydrogen bonding. A comparison of the solution structure of the APDTRP fragment with a crystal structure of the MUC1 peptide antigen bound to the breast tumor-specific antibody SM3 demonstrated significant structural similarities in the general shape.     

Nuclear magnetic resonance-based dissection of a glycosyltransferase specificity for the mucin MUC1 tandem repeat

The human glycoprotein MUC1 mucin plays a critical role in cancer progression. Breast, ovarian, and colon cancer cells often display unique cell-surface antigens corresponding to aberrantly glycosylated forms of the MUC1 tandem repeat. In this report, (15)N- and (13)C-labeled forms of a recombinant MUC1 construct containing five tandem repeats were used as substrates to define the order and kinetics of addition of N-acetylgalactosamine (GalNAc) moieties by a recombinant active form of the human enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGalNAc-T1; residues 40-559). Heteronuclear NMR experiments were performed to assign resonances associated with the two serines (Ser5 and Ser15) and three threonines (Thr6, Thr14, and Thr19) present in the 20-residue long MUC1 repeat. The kinetics and order of addition of GalNAc moieties (Tn antigen) on the MUC1 construct by human ppGalNAc-T1 were subsequently dissected by NMR spectroscopy. Threonine 14 was shown to be rapidly glycosylated by ppGalNAc-T1 with an initial rate of 25 microM/min, followed by Thr6 (8.6 microM/min). The enzyme also modified Ser5 at a slower rate (1.7 microM/min), an event that started only after the glycosylation of Thr14 and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified by ppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This study demonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominant substrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. More importantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relative simplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylation pathways and the rational design of tumor-associated MUC1 antigens.     

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.     

Mucin-type O-glycosylation and its potential use in drug and vaccine development

Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-transferases. GalNAc-transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.     

Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-Acetylgalactosamine:Peptide N-acetylgalactosaminyltransferases. site-specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions

In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitro addition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcalpha or Galbeta1-3GalNAcalpha at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position -6 (Thr-10) or -10 (Ser-6) and is inhibited by disaccharide at position -11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism.     

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.     

Structure/activity studies of the anti-MUC1 monoclonal antibody C595 and synthetic MUC1 mucin-core-related peptides and glycopeptides

MUC1 mucin is a large complex glycoprotein expressed on normal epithelial cells in humans and overexpressed and under or aberrantly glycosylated on many malignant cancer cells which consequently allows recognition of the protein core by antibodies. In order to understand how glycosylation may modulate or regulate antibody binding of mucin protein core epitopes, we have analyzed the antibody C595 (epitope RPAP) for its structure, stability, and its binding to a series of synthetic peptides and glycopeptides by a number of spectroscopic methods. Thermal and pH denaturation studies followed by changes in the CD spectrum of the antibody indicate critical involvement of specific residues to the stability of the antibody. Fluorescence binding studies indicate that alpha-N-acetylgalactosamine (GalNAc) glycosylation of a MUC1 mucin synthetic peptide TAPPAHGVT9SAPDTRPAPGS20T21APPA at threonine residues 9 and 21 and serine residue 20 enhanced the binding of antibody. The structural effects of GalNAc glycosylation on the conformation of the MUC1 peptide were studied. CD of the peptides and glycopeptides in a cryogenic mixture cooled to approximately -97 degrees C revealed that a left-handed polyproline II helix (PPII) is adopted by the peptides in solution, which appears to be further stabilized by addition of the GalNAc residues. Consistent with the PPII helical structure, which has no intra-amide hydrogen bonds, high-field NMR spectroscopy of the glycopeptide revealed no sequential dNN, medium-range, or long-range nuclear Overhauser effect (NOE) connectivities. These studies indicate that stabilization of the PPII helix by GalNAc glycosylation present the epitope of C595 antibody with a favorable conformation for binding. Furthermore, they illustrate that glycosylation of the MUC1 tumor marker protein with a simple O-linked saccharide expressed in many cancers, can enhance the binding of the clinically relevant C595 antibody.     

Mucin-type O-glycosylation and its potential use in drug and vaccine development

Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation™, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-transferases. GalNAc-transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.     

Studies of acceptor site specificities for three members of UDP-GalNAc:N-acetylgalactosaminyltransferases by using a synthetic peptide mimicking the tandem repeat of MUC5AC.

The acceptor specificity of three major isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyltranferases (murine recombinant proteins GaNTase-T1, -T2 and -T3) was investigated using the synthetic peptide (GTTPSPVPTTSTTSAP) containing clusters of threonine residues mimicking the mucin tandem repeat unit of MUC5AC. The O-glycosylated products obtained after in vitro reactions were fractionated by capillary electrophoresis and the purified glycopeptides were characterized by MALDI mass spectrometry (number of O-GalNAc residues) and by Edman degradation (site location). A maximum of three GalNAc residues was transferred into the MUC5AC motif peptide and the preferential order of incorporation for each GaNTase isoform was determined. Our results suggest that clusters of threonine appear to be essential for site recognition of peptide backbone by the ubiquitous GaNTases and also support the notion that the different GaNTase isoforms with varying substrate specificities are involved in a hierarchical order of O-glycosylation processing of the mucin-type O-glycoproteins.     

Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions

MUC1 mucin is a large transmembrane glycoprotein, of which the extracellular domain is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is underglycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above) as well as the normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc), and TF (Gal beta1-3 GalNAc) carbohydrates. In the present study, NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRP core epitope region to the recognition and binding of a monoclonal antibody, Mab B27.29, raised against the intact tumor-associated MUC1 mucin. Four peptides were studied: a MUC1 16mer peptide of the sequence Gly1-Val2-Thr3-Ser4-Ala5-Pro6-Asp7-Thr8-Arg9-Pro10-Ala11-Pro12-Gly13-Ser14-Thr15-Ala16, two singly Tn-glycosylated versions of this peptide at either Thr3 or Ser4, and a doubly Tn-glycosylated version at both Thr3 and Ser4. The results of these studies showed that the B27.29 MUC1 B-cell epitope maps to two separate parts of the glycopeptide, the core peptide epitope spanning the PDTRP sequence and a second (carbohydrate) epitope comprised of the Tn moieties attached at Thr3 and Ser4. The implications of these results are discussed within the framework of developing a glycosylated second-generation MUC1 glycopeptide vaccine.     

Effects of glycosylation on the conformation and dynamics of O-linked glycoproteins: carbon-13 NMR studies of ovine submaxillary mucin

Carbon-13 NMR spectroscopic studies of native and sequentially deglycosylated ovine submaxillary mucin (OSM) have been performed to examine the effects of glycosylation on the conformation and dynamics of the peptide core of O-linked glycoproteins. OSM is a large nonglobular glycoprotein in which nearly one-third of the amino acid residues are Ser and Thr which are glycosylated by the alpha-Neu-NAc(2-6)alpha-GalNAc- disaccharide. The beta-carbon resonances of glycosylated Ser and Thr residues in intact and asialo mucin display considerable chemical shift heterogeneity which, upon the complete removal of carbohydrate, coalesces to single sharp resonances. This chemical shift heterogeneity is due to peptide sequence variability and is proposed to reflect the presence of sequence-dependent conformations of the peptide core. These different conformations are thought to be determined by steric interactions of the GalNAc residue with adjacent peptide residues. The absence of chemical shift heterogeneity in apo mucin is taken to indicate a loss in the peptide-carbohydrate steric interactions, consistent with a more relaxed random coiled structure. On the basis of the 13C relaxation behavior (T1 and NOE) the dynamics of the alpha-carbons appear to be unique to each amino acid type and glycosylation state, with alpha-carbon mobilities decreasing in the order Gly greater than Ala = Ser greater than Thr much greater than monoglycosylated Ser/Thr approximately greater than disaccharide linked Ser/Thr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural features of the human salivary mucin, MUC7

Human salivary mucin (MUC7) is characterized by a single polypeptide chain of 357 aa. Detailed analysis of the derived MUC7 peptide sequence reveals five distinct regions or domains: (1) an N-terminal basic, histatin-like domain which has a leucine-zipper segment, (2) a moderately glycosylated domain, (3) six heavily glycosylated tandem repeats each consisting of 23 aa, (4) another heavily glycosylated MUC1- and MUC2-like domain, and (5) a C-terminal leucine-zipper segment. Chemical analysis and semi-empirical prediction algorithms for O-glycosylation suggested that 86/105 (83%) Ser/Thr residues were O-glycosylated with the majority located in the tandem repeats. The high (～25%) proline content of MUC7 including 19 diproline segments suggested the presence of polyproline type structures. CD studies of natural and synthetic diproline-rich peptides and glycopeptides indicated that polyproline type structures do play a significant role in the conformational dynamics of MUC7. In addition, crystal structure analysis of a synthetic diproline segment (Boc-Ala-Pro-OBzl) revealed a polyproline type II extended structure. Collectively, the data indicate that the polyproline type II structure, dispersed throughout the tandem repeats, may impart a stiffening of the backbone and could act in consort with the glycosylated segments to keep MUC7 in a semi-rigid, rod shaped conformation resembling a ‘bottle-brush’ model.     

Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases

Mammalian mucin-type O-glycosylation is initiated by a large family of ～20 UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer α-GalNAc from UDP-GalNAc to Ser and Thr residues of polypeptide acceptors. Characterizing the peptide substrate specificity of each isoform is critical to understanding their properties, biological roles, and significance. Presently, only the specificities of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 have been systematically characterized utilizing random peptide substrates. We now extend these studies to ppGalNAc T3, T5, and T12, transferases variously associated with human disease. Our results reveal several common features; the most striking is the similar pattern of enhancements for the three residues C-terminal to the site of glycosylation for those transferases that contain a common conserved Trp. In contrast, residues N-terminal to the site of glycosylation show a wide range of isoform-specific enhancements, with elevated preferences for Pro, Val, and Tyr being the most common at the -1 position. Further analysis reveals that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue enhancements varied among transferases, thus further modulating substrate preference in an isoform-specific manner. By utilizing the obtained transferase-specific preferences, the glycosylation patterns of the ppGalNAc Ts against a series of peptide substrates could roughly be reproduced, demonstrating the potential for predicting isoform-specific glycosylation. We conclude that each ppGalNAc T isoform may be uniquely sensitive to peptide sequence and overall charge, which together dictates the substrate sites that will be glycosylated.     

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O‐glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O‐glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn‐antigen (GalNAc‐O‐Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding‐competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution‐state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre‐selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.     

Conservation of peptide acceptor preferences between Drosophila and mammalian polypeptide-GalNAc transferase ortholog pairs

UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltrans- ferases (ppGalNAc Ts) comprise a large family of glycosyltransferases that initiate mucin-type protein O-glycosylation, transferring alpha-GalNAc to Thr and Ser residues of polypeptide acceptors. Families of ppGalNAc Ts are found across diverse eukaryotes with orthologs identifiable from mammals to single-cell organisms. The peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly understood. Previously, we reported a series of oriented random peptide substrate libraries for quantitatively determining the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 (Gerken TA, Raman J, Fritz TA, Jamison O. 2006. Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 & T2 (ppGalNAc T1 & T2) derived from oriented random peptide substrates. J Biol Chem. 281:32403-32416). With these substrates, previously unknown features of the transferases were revealed. Utilizing these and a new lengthened set of random peptides, studies have now been performed on PGANT5 and PGANT2, the Drosophila orthologs of T1 and T2. The results from these studies suggest that the major peptide substrate determinants for these transferases are contained within 2 to 3 residues flanking the site of glycosylation. It is further found that the mammalian and fly T1 orthologs display very similar peptide substrate preferences, while the T2 orthologs are nearly indistinguishable, suggesting similar peptide preferences amongst orthologous pairs have been maintained across evolution. This conclusion is further supported by sequence homology comparisons of each of the transferase orthologs, showing that the peptide substrate and UDP binding site residues are more highly conserved between species relative to their remaining catalytic and lectin domain residues.     

Recombinant human tumor antigen MUC1 expressed in insect cells: structure and immunogenicity

MUC1, a member of the mucin family of molecules, is a transmembrane glycoprotein abundantly expressed on human ductal epithelial cells and tumors originating from those cells. MUC1 expressed by malignant cells is aberrantly O-glycosylated. Differences in O-glycosylation of the tandem repeat region of MUC1 make tumor and normal forms of this antigen immunologically distinct. The tumor-specific glycoform is, therefore, expected to be a good target for immunotherapy and a good immunogen for generation of antitumor immune responses. We have generated a renewable source of this glycoform by expressing MUC1 cDNA in Sf-9 insect cells using a baculovirus vector. This form of MUC1 (BV-MUC1) is O-glycosylated at a very low level, approximately 0.3% (w/w), and this is not due to the lack of appropriate glycosylotransferases in insect cells. Peptidyl GalNAc-transferases isolated from Sf-9 cells were able to glycosylate in vitro a synthetic MUC1 peptide as efficiently as the transferases isolated from human milk. Neither preparation of peptidyl GalNAc-transferases, however, was able to glycosylate BV-MUC1. This underglycosylated recombinant MUC1 mimics underglycosylated MUC1 on human tumor cells and could serve as an immunogen to stimulate responses that would recognize MUC1 on tumor cells. To test this we immunized mice with Sf-9 cells expressing BV-MUC1. Sera from immunized mice recognized MUC1 on human tumor cells. We also generated MUC1-specific T cells that proliferated in response to synthetic MUC1 peptide.     

Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity

UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence specificity, whereas the primary function of the lectin domain is to increase affinity to previously glycosylated substrates. Whether the lectin domain also has peptide sequence selectivity has remained unclear. Using a glycopeptide array with a library of synthetic and recombinant glycopeptides based on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate an additional level of complexity in the initiation step of O-glycosylation by GalNAc-Ts.     

Polypeptide GalNAc-transferases, ST6GalNAc-transferase I, and ST3Gal-transferase I expression in gastric carcinoma cell lines.

Mucin O-glycosylation in cancer is characterized by aberrant expression of immature carbohydrate structures leading to exposure of simple mucin-type carbohydrate antigens and peptide epitopes. Glycosyltransferases controlling the initial steps of mucin O-glycosylation are responsible for the altered glycosylation observed in cancer. We studied the expression in gastric cell lines of six UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T1, T2, T3, T4, T6, T11) that catalyze the initial key step in the regulation of mucin O-glycosylation, the transfer of GalNAc from UDP-GalNAc to serine and threonine residues. We also studied the expression of ST6GalNAc-I, the enzyme responsible for the synthesis of Sialyl-Tn antigen (NeuAcalpha2,6GalNAc) and the ST3Gal-I, the enzyme responsible for the synthesis of Sialyl-T antigen (NeuAcalpha2,3Galbeta1,3GalNAc). This study was done using specific monoclonal antibodies, enzymatic assays, and RT-PCR. Our results showed that GalNAc-T1, -T2, and -T3 have an ubiquitous expression in all gastric cell lines, whereas GalNAc-T4, -T6, and -T11 show a restricted expression pattern. The immunoreactivity with MAb VU-2-G7 suggests that, apart from GalNAc-T4, another GalNAc transferase is involved in the glycosylation of the Thr in the PDTR region of the MUC1 tandem repeat. The expression of ST3Gal-I correlates with the expression of the Sialyl-T antigen in gastric cell lines and in the control cell lines studied. The expression of ST6GalNAc-I is low in gastric cell lines, in accordance with the low/absent expression of the Sialyl-Tn antigen.     

Mucin-1-related T cell infiltration in colorectal carcinoma

 Mucins (MUC) are highly glycosylated molecules widely expressed on epithelia of different origins, including colonic mucosa. Altered glycosylation processes in tumour cells result in the exposure of normally cryptic peptide epitopes, which may then be recognized as tumour-specific antigens. Recently, MUC1-specific antibodies were detected in the serum of a broad range of cancer patients, and from different tumours tumour-specific cytotoxic T lymphocytes (CTL) were isolated that recognized MUC1. Absence of HLA restriction in the recognition has been ascribed to the highly repetitive sequence of the polypeptide core, allowing simultaneous recognition of multiple identical epitopes and cross-linking and aggregation of T cell receptor on mucin-specific T cells. We investigated the expression of MUC1 epitopes in 56 cell suspensions from Dukes’ B to D colorectal carcinomas using antibodies that recognize distinct peptide sequences on the glycosylated or deglycosylated MUC1 protein backbone. No relation was observed between MUC1 expression, or the extent of its glycosylation, and Dukes’ stage, tumour location and tumour differentiation, but a positive correlation was detected between the percentages of tumour cells expressing mucin-1 and the numbers of CD3⁺ infiltrating cells. These tumour-infiltrating lymphocytes contained, however, only a few MUC1-specific T lymphocytes, as CTL showing preferential killing of MUC1-expressing target cells were only obtained from one tumour. Since, in addition, the majority of colorectal carcinomas were found to express the fully glycosylated MUC1 glycoprotein, its potential role as a target antigen for T-lymphocyte-mediated immunotherapy in this tumour type is probably limited. Received: 2 April 1996 / Accepted: 28 May 1996