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.     

Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.     

Structural and ICAM-1-Docking Properties of a Cyclic Peptide from the I-domain of LFA-1: An inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion

Abstract

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)- Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7-Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 α-subunit. We found that cyclic peptide 1 can bind to the D1- domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1- domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable βII'-turn at Asp4-Gly5-Glu6-Ala7 and a βI-turn at Pen1-Ile2-Thr3-Asp4; a less stable βV-turn is found at the C-terminal region. The β-turn at Asp4-Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1- domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Solution structure of a peptide derived from the beta subunit of LFA-1

Cell-adhesion molecules are critical for immune response. It is well known that the inhibition of adhesion is very effective in immunotherapy and that the peptides derived from leukocyte function associated antigen (LFA-1) and intercellular adhesion molecule (ICAM-1) modulate cell-adhesion interaction. The three-dimensional structure of a cyclic peptide, Cyclo(1,12)Pen(1)-Asp(2)-Leu(3)-Ser(4)-Tyr(5)-Ser(6)-Leu(7)-Asp(8)-Asp(9)-Leu(10)-Arg(11)-Cys(12) (cLBEL) derived from the beta subunit of LFA-1 which is known to modulate homotypic T-cell-adhesion process has been studied using NMR, CD and molecular dynamics (MD) simulation. The peptide exhibits two possible conformations in solution. Structure I has a conformation with two consecutive beta-turns involving residues Tyr(5)-Ser(6)-Leu(7)-Asp(8) and Asp(9)-Leu(10)-Arg(11)-Cys(12). Structure II has a beta-turn at Tyr(5)-Ser(6)-Leu(7)-Asp(8) and forms a beta-hairpin type of conformation.     

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.     

Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study

MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.     

Binding characteristics of an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132).

To determine the epitopic structure for an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, a major glycopeptide designated as T3 was obtained as an immunoaffinity column-bound fraction. On solid-phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treatment with thermolysin, T3 was converted into about 50 fragments, as found on fractionation by HPLC. Several of them were strongly immunoreactive and had the same amino acid sequence, i.e. Phe-Ser*-Gly-Glu-Thr*-Ser*-Thr*-Thr*-Val-Ile-Ser*-Gly-Thr*-Asn-Val, where asterisks denote the sites of attachment of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of the carbohydrate attached in these glycopeptides led us to postulate that a cluster composed of four sialyl Tn antigens is the essential epitopic structure for MLS 132.     

Bradykinin analogs containing the 4-amino-2-benzazepin-3-one scaffold at the C-terminus.

High affinity peptide ligands for the bradykinin (BK) B(2) subtype receptor have been shown to adopt a beta-turn conformation of the C-terminal tetrapeptide (H-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH). We investigated the replacement of the Pro(7)-Phe(8) dipeptide moiety in BK or the D-Tic(7)-Oic(8) subunit in HOE140 (H-D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-Oic(8)-Arg(9)-OH) by 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one templates (Aba). Binding studies to the human B(2) receptor showed a correlation between the affinities of the BK analogs and the propensity of the templates to adopt a beta-turn conformation. The L-spiro-Aba-Gly containing HOE140 analog BK10 has the best affinity, which correlates with the known turn-inducing property of this template. All the compounds did not modify basal inositolphosphate (IP) output in B(2)-expressing CHO cells up to 10 microM concentration. The antagonist properties were confirmed by the guinea pig ileum smooth muscle contractility assay. The new amino-benzazepinone (Aba) substituted BK analogs were found to be surmountable antagonists.     

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.     

Conformational studies of two bradykinin antagonists by using two-dimensional NMR techniques and molecular dynamics simulations

The solution conformations of two potent antagonists of bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), [Aca(-1),DArg0,Hyp3,Thi5,DPhe7,(N-Bzl)Gly8]BK (1) and [Aaa(-1),DArg0,Hyp3,Thi5,(2-DNal)7,Thi8]BK (2), were studied by using 2D NMR spectroscopy in DMSO-d6 and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe7-(N-Bzl)Gly8 peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a beta-turn structure for the Hyp3-Gly4 residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe7 and (N-Bzl)Gly8 in analogue 1 suggests type VI beta-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb beta-turn comprising residues Ser6-Arg9 and the betaI or betaII-turn involving the Pro2-Thi5 fragment, whereas peptide 2 shows the tendency towards the formation of type I beta-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9. Moreover, the side chain of DArg0 is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

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.     

Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128).

To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc)-Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies.     

Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat

The cell membrane mucin MUC1 is over-expressed and aberrantly glycosylated in many cancers, and cancer-associated MUC1 glycoforms represent potential targets for immunodiagnostic and therapeutic measures. We have recently shown that MUC1 with GalNAcalpha1-O-Ser/Thr (Tn) and NeuAcalpha2-6GalNAcalpha1-O-Ser/Thr (STn) O-glycosylation is a cancer-specific glycoform, and that Tn/STn-MUC1 glycopeptide-based vaccines can override tolerance in human MUC1 transgenic mice and induce humoral immunity with high specificity for MUC1 cancer-specific glycoforms (Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, et al. 2006. Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology. 16:96-107). In order to further characterize the immune response to Tn/STn-MUC1 glycoforms, we generated monoclonal antibodies with specificity similar to the polyclonal antibody response found in transgenic mice. In the present study, we define the immunodominant epitope on Tn/STn-MUC1 glycopeptides to the region including the amino acids GSTA of the MUC1 20-amino acid tandem repeat (HGVTSAPDTRPAPGSTAPPA). Most other MUC1 antibodies are directed to the PDTR region, although patients with antibodies to the GSTA region have been identified. A panel of other MUC1 glycoform-specific monoclonal antibodies was included for comparison. The study demonstrates that the GSTA region of the MUC1 tandem repeat contains a highly immunodominant epitope when presented with immature short O-glycans. The cancer-specific expression of this glycopeptide epitope makes it a prime candidate for immunodiagnostic and therapeutic measures.     

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.     

'Wave-type' structure of a synthetic hexaglycosylated decapeptide: A part of the extracellular domain of human glycophorin A

The three-dimensional structure of a glycopeptide, His-Thr*-Ser*-Thr*-Ser*-Ser*-Ser*-Val-Thr-Lys, with 2-acetamido-2-deoxy--D-galactose (GalNAc) residues linked to six adjacent amino acids from Thr-10 to Ser-15, was studied by NMR spectroscopy and molecular dynamics (MD) simulations. The hexaglycosylated decapeptide is part of the extracellular domain of human glycophorin A and shows an extended structure of the peptide backbone due to O-glycosylation. Furthermore, each GalNAc residue exhibits one and only one NOE contact from the NHAc proton to the backbone amide proton of the amino acid that the sugar is directly bound to. This indicates a strong preference for the orientation of all GalNAc residues towards the N-terminus. NOE build-up curves were used to determine 42 inter-proton distances that, in connection with angles of the peptide backbone obtained from 3J-coupling constants, resulted in constraints for a MD simulation in water. The NMR data and the MD simulations show a preference for an extended backbone structure. The GalNAc residues are located alternatingly on opposite sides of the backbone and reduce the flexibility of the peptide backbone. The conformation of the molecule is relatively rigid and shows a 'wave-type' 3D structure of the peptide backbone within the glycosylation cluster. This new structural element is also supported by the unusual CD spectrum of the glycopeptide.