A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats

The nucleotide sequences of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin were determined, and a large domain was found to consist of a 60-base pair tandem repeat sequence. The cDNA clones were originally selected (Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., and Taylor-Papadimitriou, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6060-6064) using three monoclonal antibodies which show differential reactivity with the mucin produced by normal and malignant breast. Two of the epitopes are exposed in the normally processed and cancer-associated mucin, while one epitope is unmasked only in the cancer-associated mucin (Burchell, J. M., Durbin, H., and Taylor-Papadimitriou, J. (1983) J. Immunol. 131, 508-513; Burchell, J., Gendler, S., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. (1987) Cancer Res. 47, 5476-5482). We show here that all three antibodies react with a synthetic peptide with an amino acid sequence corresponding to that predicted by the tandem repeat. Identification of the epitopes preferentially expressed on the cancer-associated mucin should allow a directed approach to the development of tumor-specific antibodies using synthetic peptides as immunogens.     

Heterogeneity in the protein cores of mucins isolated from human middle ear effusions: evidence for expression of different mucin gene products

High molecular weight mucins were isolated and purified from human middle ear effusions of children with Otitis Media with Effusion (OME) classified into three groups, (1) thick and (2) thin from anatomically normal children and (3) effusions from cleft palate patients. Amino acid analyses of the purified mucins from the three pools were similar but not identical with characteristic contents of serine threonine and proline (32%, 28%, and 38% for pools (1) (2) and (3) respectively). Proteinase resistant glycopeptide fragments corresponding to the tandem repeat domains of cloned mucin genes showed marked differences both between the three mucin pools and with the composition of the tandem repeat sequences of the cloned mucin genes expressed in the airways. Studies on the antigenic identity of middle ear mucins found an epitope likely to be present on MUC5AC, but only accounting for a maximum of 15% by weight and no reactivity was found with antibodies to MUC2 or MUC1. A polyclonal antibody raised to thick effusion mucins reacted strongly with human salivary mucin suggesting the presence of MUC5B epitopes. These studies suggest that more than one mucin gene product is secreted by the human middle ear mucosa and that there may be further mucin genes expressed by the middle ear that have yet to be cloned.     

Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUC1 mucin gene: Effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUC1 gene

The immune response of CAF1 mice to various synthetic peptides (SP) related to the amino acid sequence (PDTRPAPGSTAPPAHGVTSA) of the tandem repeat of the MUC1 human breast mucin core peptide was evaluated. The most immunogenic preparations of the synthetic peptides were those conjugated to keyhole limpet hemocyanin (KLH) or clustered in a dendritic multiple antigenic peptide (MAP-4) configuration. The mice were immunized subcutaneously with synthetic peptides emulsified in RIBI adjuvant, employing various immunization protocols. Equivalently high IgG responses were induced using SP-KLH conjugates (GVTSAPDTRPAPGSTA-KLH) or an SP — MAP-4 chimeric configuration (SP1-6), which also included a universal malarial CST-3 T-helper epitope (SP1-6 = SAPDTRPAEKKIAKMEKASSVFNVVNS — MAP-4). These IgG antibodies bound both the appropriate MUC1 synthetic peptides and the cell surface expressed MUC1 mucin on murine mammary cells that had been transfected with the human MUC1 gene and a human breast cancer cell line that expresses cell-surface MUC1. A MAP-4 molecule, which included the entire 20-aminoacid sequence of the MUC1 tandem repeat (SP1-5 = PDTRPAPGSTAPPAHGVTSA—MAP-4) induced a poor IgG response. In contrast, all three types of molecule: SP-KLH, SP1-6 and SP1-5, were found to be good immunogens for the induction of specific delayedtype hypersensitivity (DTH) reactions measured using either synthetic peptides or MUC1-transfected cells. In addition, immunization with irradiated MUC1-transfected cells induced strong DTH reactions measured using synthetic peptides that expressed the PDTRP sequence, which has been shown to be, or to overlap, a T cell epitope in humans and a B cell epitope in mice. Finally, it was demonstrated that synthetic MUC1 peptide vaccines could be used both prophylactically and therapeutically to inhibit the growth of MUC1-transfected tumor cells and prolong the survival of tumor-bearing mice.     

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50–200 by in length and cloning these into the 5 terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.     

Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice

Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species.     

Cloning and sequencing of a human pancreatic tumor mucin cDNA

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.     

High-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus-based vector

Porcine epidemic diarrhea virus (PEDV) causes acute enteritis in pigs of all ages and is often fatal for neonates. A tobacco mosaic virus (TMV)-based vector was utilized for the expression of a core neutralizing epitope of PEDV (COE) for the development of a plant-based vaccine. In this study, the coding sequence of a COE gene was optimized based on the modification of codon usage in tobacco plant genes and the removal of mRNA-destabilizing sequences. The native and synthetic COE genes were cloned into TMV-based vectors and expressed in tobacco plants. The recombinant COE protein constituted up to 5.0% of the total soluble protein in the leaves of tobacco plants infected with the TMV-based vector containing synthetic COE gene, which was approximately 30-fold higher than that in tobacco plants infected with TMV-based vector containing a native COE gene. Therefore, this result indicates that the plant viral expression system with a synthetic gene optimized for plant expression is suitable to produce a large amount of antigen for the development of plant-based vaccine rapidly.     

Alkali-catalyzed β-elimination of periodate-oxidized glycans: A novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections

Altered expression of mucin gene products has been described in many epithelial cancers including colorectal cancer. However, mucins are heavily O-glycosylated making the study of apomucin expression difficult. In this study, we describe a novel method of chemical deglycosylation of mucin gene products on paraffin embedded formalin-fixed tissue sections. In the normal and cancerous colorectum, our results suggest that alkali-catalyzed -elimination of periodate oxidized glycan method of chemical deglycosylation modifies the structure of carbohydrates sensitive to mild periodate oxidation resulting in less steric hindrance and selectively removes Tn and sialyl-Tn structures, partially exposing the underlying apomucin epitopes. Using this method, we have demonstrated that the MUC1 tandem repeat epitope recognized by MAb 139H2 is masked predominantly due to steric hindrance by carbohydrate structures whereas the MUC2 tandem repeat epitope recognized by MAb CCP58 and pAb MRP and the MUC3 tandem repeat epitope recognized by pAb M3P are masked by the presence of carbohydrate side chains O-linked to Ser/Thr residues within the epitope. Considerable differences in the level and pattern of expression of the epitopes in the tandem repeat region of apomucins of MUC1, MUC2, and MUC3 were observed between normal and cancerous colorectal cancer tissues. We conclude that this novel chemical deglycosylation method that causes selective cleavage of distinct glycans will be useful in unmasking various mucin gene products and glycoproteins containing similar O-glycosidic linkages in the tissue sections of formalin-fixed paraffin embedded normal and pathological tissues.     

Localisation of a protein core-specific epitope from gastrointestinal mucin (MUC2). The effect of epitope immobilisation on antibody recognition

Human intestinal mucins are high molecular weight glycoproteins which protect and lubricate the epithelium of the gastrointestinal tract. In cases of malignant disease, mucins are abnormally expressed, overproduced or underglycosylated. This feature may enable the mucins to serve as tumour markers. The MUC2 mucin largely consists of a variable number of tandem repeats of a 23 amino acid sequence, ¹PTTTPITTTTTVTPTPTPTGTQT²³. In this study we have localised the minimal and the optimal epitope within this region by the previously developed protein core specific 996 monoclonal antibody using synthetic peptides. Several overlapping and truncated peptides related to the tandem repeat unit have been prepared by solid-phase methodology. Other mucin peptides were synthesised on the tips of polyethylene pins, and these remained C-terminally attached to the pins for comparative investigations. The interaction of the 996 monoclonal antibody with the synthetic peptides was studied either in solution by competition RIA or on immobilised peptides by indirect ELISA experiments. These experiments show that the minimal epitope recognised by the 996 antibody is the Ac-¹⁹TGTQ²² (IC₅₀=3100 μm in solution). For the optimal 996 antibody binding in solution the ¹⁶PTPTGTQ²² heptapeptide (IC₅₀=3 μm ) is required. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Production of MUC1 and MUC2 mucins by human tumor cell lines.

A mucus secreting, clonal derivative (HT29-SB) of the human colonic adenocarcinoma cell line HT29, and the LS174T colon cancer cell line, secrete mucin into the culture medium as a viscoelastic gel. Mab BC2, which defines a peptide epitope present in the variable number of tandem repeats (VNTR) of the MUC1 core protein, reacted with this material after deglycosylation. Two high molecular weight bands were detected in TFMSA treated gel-formed mucin from HT29-SB and LS174T by western blotting (Mr 580 kDa and 420 kDa). A similar pattern of reactivity was seen with the culture supernatants from HT29-SB, the ovarian tumor cell line COLO-316, and the breast cancer cell line MCF-7. Mab CCP58 (anti-MUC2 VNTR) reacted with a 580 kDa band in gel-formed mucin produced by LS174T, but was not reactive with mucin produced by the other cell lines. The findings indicate that human colonic cell lines, in addition to breast and ovarian cell lines, may both express and secrete the MUC1 protein core, and that the LS174T cell line expresses and secretes both the MUC1 and MUC2 core proteins.     

Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism.

We present here the full-length cDNA sequence and genomic structure of the mouse homologue of the tumor-associated mucin, MUC1. This mucin (previously called polymorphic epithelial mucin) is present at the apical surface of most glandular epithelial cells. The mouse gene, Muc-1, encodes an integral membrane protein with 40% of its coding capacity made up of serine, threonine, and proline, a composition typical of a highly O-glycosylated protein. The mucin core protein consists of an amino-terminal signal sequence, a tandem repeat domain encoding 16 repeats of 20-21 amino acids, and unique sequence containing transmembrane and cytoplasmic domains. Homology with the human protein is only 34% in the tandem repeat domain, mainly showing conservation of serines and threonines, presumed sites of O-linked carbohydrate attachment. Homology rises to 87% in the transmembrane and cytoplasmic domains, suggesting that these regions may be functionally important. The pattern of expression of the mouse mucin is very similar to that of its human counterpart and accordingly the two promoter regions share high homology, 74%, although previously identified potential hormone-responsive elements are not conserved. Interestingly, the mouse homologue, unlike its human counterpart does not exhibit a variable number tandem repeat polymorphism. We present evidence that suggests that the mouse gene was at one time polymorphic but has mutated away from this state.     

Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide

Type II collagen (CII) in joint cartilage is known to be a major auto-antigen in human rheumatoid arthritis. Several animal model- and clinical-studies on tolerance-based immunotherapy for the arthritis have been conducted by administrating synthetic immunodominant peptides through an oral route. In the present study, to produce a tolerogenic peptide with therapeutic potential in transgenic rice plants, a gene construct producing glutelin fusion protein with tandem four repeats of a CII_250–270 peptide (residues 250–270) (GluA-4XCII_250–270) containing a human T-cell epitope was introduced with a selection marker, hygromycin phosphotransferase gene (hygromycin-resistance gene) (hph), by co-transformation. Several transgenic plants with high and stable expression of gluA-4XCII _250–270 , but no hph, were selected based on both DNA and protein analyses. The GluA-4XCII_250–270 fusion proteins were detected as both precursor and processed forms mainly in a glutelin fraction of rice endosperm protein extracts and in protein-body rich fractions prepared by density gradient ultracentrifugation. The amount of accumulated CII_250–270 peptide was immunochemically estimated to be about 1 μg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 μg of the peptide per mouse a day) for 2 weeks showed tendencies lowering and delaying serum specific-IgG2a response against subsequent and repeated intraperitoneal-injection of type II collagen. Taken these together, the CII-immunodominant peptide could effectively be produced and accumulated as a glutelin-fusion protein in the transgenic rice seeds, which might be useful as pharmaceutical materials and functional food for prevention and therapy for anti-CII autoimmune diseases like human rheumatoid arthritis.     

Inhibitorily active recombinant human stefin B. Gene synthesis, expression and isolation of an inhibitory active MS-2 pol-stefin B fusion protein and preparation of Des[Met1,2(2)]stefin B

A synthetic gene coding for the human intracellular cysteine proteinase inhibitor, stefin B, was constructed from 13 chemically synthesized oligonucleotides according to the method of Khorana. The gene was inserted into the plasmid vector pTZ, amplified and sequenced. For expression, a temperature-inducible system producing fusion proteins was used. With the vector pEx31A containing the synthetic cystatin B gene, E. coli strain 537 produced a fusion protein of the N-terminal part of bacteriophage MS-2 polymerase and [Met-2Gly-1]stefin B. Lysates of the induced bacteria were inhibitorily active against papain. The fusion protein was expressed in high yield (about 20% of total E. coli proteins) and mostly deposited as inclusion bodies. The unfolded fusion protein was partially purified in the presence of urea. After refolding, approx. 6% of the protein was inhibitorily active against papain, human cathepsin H and B. Des[Met1,2(2)]stefin B was released by cyanogen bromide cleavage of the fusion protein and identified by N-terminal amino-acid sequence analysis. The non-separated cleavage products were also inhibitorily active after refolding. The estimated inhibition constants for the fusion protein and its cleavage products were similar to those reported for natural stefin B.     

Human antibodies react with an epitope of the human papillomavirus type 6b L1 open reading frame which is distinct from the type-common epitope.

Recombinant proteins encoded by the human papillomavirus type 6b (HPV6b) L1 open reading frame react with sera from patients with condylomata acuminata and also react with rabbit antiserum raised against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV1) virions. To map the immunoreactive epitopes, a series of procaryotic expression plasmids was made which contained a nested set of 3' to 5' deletions in the HPV6b L1 open reading frame. The deleted plasmids expressed a set of carboxy to amino terminus truncated fusion proteins. Regions containing the immunoreactive epitopes were mapped by determining which of the deleted fusion proteins retained reactivity with sera in Western immunoblot assays. The coding sequence for a human antibody-reactive linear epitope mapped between HPV6b nucleotide coordinates 7045 and 7087, and the rabbit anti-BPV1-reactive epitope coding sequence mapped between coordinates 6377 and 6454. Synthetic peptides derived from the epitope mapping were reacted with sera in enzyme-linked immunosorbent assay. Human sera reacted with synthetic peptide QSQAITCQKPTPEKEKPDPYK (HPV6b L1 amino acids 417 through 437). Rabbit anti-BPV1 and rabbit antisera raised against HPV16 L1 recombinant proteins reacted with the synthetic peptide DGDMVDTGFGAMNFADLQTNKSDVPIDI (HPV6b L1 amino acids 193 through 220). Human sera which reacted with HPV6b L1 fusion proteins cross-reacted with an HPV11 L1 fusion protein but did not react with fusion proteins encoded by HPV1a, HPV16, or HPV18. Rabbit anti-BPV1 reacted with L1 fusion proteins encoded by all of these HPV types. In contrast to the type-common (rabbit anti-BPV1-reactive) epitope, the human antibody-reactive epitope appears to be relatively HPV type specific.     

A new class of receptor for herpes simplex virus has heptad repeat motifs that are common to membrane fusion proteins

We isolated a human cDNA by expression cloning and characterized its gene product as a new human protein that enables entry and infection of herpes simplex virus (HSV). The gene, designated hfl-B5, encodes a type II cell surface membrane protein, B5, that is broadly expressed in human primary tissue and cell lines. It contains a high-scoring heptad repeat at the extracellular C terminus that is predicted to form an alpha-helix for coiled coils like those in cellular SNAREs or in some viral fusion proteins. A synthetic 30-mer peptide that has the same sequence as the heptad repeat alpha-helix blocks HSV infection of B5-expressing porcine cells and human HEp-2 cells. Transient expression of human B5 in HEp-2 cells results in increased polykarocyte formation even in the absence of viral proteins. The B5 protein fulfills all criteria as a receptor or coreceptor for HSV entry. Use by HSV of a human cellular receptor, such as B5, that contains putative membrane fusion domains provides an example where a pathogenic virus with broad tropism has usurped a widely expressed cellular protein to function in infection at events that lead to membrane fusion.     

Identification of the 48-kDa G11 protein from guinea pig testes as sperad

A protein found specifically in the membrane of spermatozoa called G11 has been implicated in sperm-egg binding and fusion. This study describes purification and identification of the G11 antigen. The G11 protein was purified using anion exchange chromatography, immunoaffinity chromatography and preparative SDS-PAGE and was subjected to amino acid microsequencing by tandem mass spectrometry. Internal amino acid sequence data derived from the 48-kDa G11 protein revealed that G11 is the recently discovered guinea pig sperm protein, sperad. Sperad is a transmembrane protein present in the periacrosomal plasma membrane of guinea pig sperm. The cytoplasmic domain of sperad was amplified from a guinea pig testes cDNA expression library by polymerase chain reaction and cloned into a prokaryotic gene expression vector, pGEX-2T. The recombinant glutathione S-transferase fusion protein was immunoblotted with monoclonal antibody G11. The results obtained from this study confirmed the monoclonal antibody G11 epitope location on the cytoplasmic domain of sperad.     

Stoichiometry of murine leukemia virus envelope protein-mediated fusion and its neutralization

Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.