MUC4 is a major component of salivary mucin MG1 secreted by the human submandibular gland

High molecular weight salivary mucin (MG1) is an important component of saliva, contributing to the lubricative and tissue-protective functions of this biological fluid. We have shown previously that the human mucin gene MUC5B is expressed at high levels in sublingual gland and is a significant constituent of MG1. Since many epithelia express multiple mucin genes, it seemed likely that MG1 in salivary secretions is also a heterogeneous mixture of mucin gene products. The aim of this study was to determine whether MUC4, a mucin shown in Northern blotting experiments to be expressed in salivary glands, was a significant protein component of MG1 in salivary secretions. Two cDNA clones containing MUC4 tandem repeats were isolated from a human submandibular gland cDNA library. In addition, recombinant MUC4 produced in a bacterial expression system cross-reacted with an antibody directed against deglycosylated MG1. This shows conclusively that human salivary mucin MG1 contains both MUC5B and MUC4 gene products suggesting that each mucin may perform distinct functions in the oral cavity.     

Analysis of Candida albicans adhesion to salivary mucin

Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.     

Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7)

Previous biochemical studies have determined that human saliva contains high and low molecular weight mucin glycoproteins (MG1 and MG2, respectively) that are structurally distinct. In this study, we describe the isolation and characterization of overlapping cDNA clones which code for the MG2 protein core. DNA sequencing revealed a translated region of 1131 nucleotides encoding a protein of 377 amino acid residues with a molecular mass of 39 kDa. The first 20 N-terminal residues were very hydrophobic and probably comprise the MG2 leader peptide. The region encoding the secreted protein can be divided into three distinct domains; unique 5'- and 3'-translated regions containing 4 and 1 potential N-glycosylation sites, respectively, and a central region of six almost perfect tandem repeats of 23 amino acid residues with a high number of Thr and Ser. No sequence homology with any other human or animal mucins, and no significant homology to any other proteins was found. MG2 mRNA is about 2.5 kilobases long, and its expression appears to be species-, tissue-, and cell-specific. We propose to name this gene MUC7 in accordance with the mucin genes cloned to date named MUC1-MUC6.     

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression

BACKGROUND & AIMS: Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS: Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS: Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunner's glands of the duodenum. CONCLUSIONS: MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.     

A novel gastric-cancer-associated mucin antigen defined by a monoclonal antibody A3D4

De-glycosylation of mucins may expose new tumor-associated core protein epitopes. In this study, to attempt to develop useful markers for gastric cancers, we have purified and de-glycosylated gastric mucin and tried to establish monoclonal antibodies (MAbs). A MAb designated A3D4 among established MAbs was shown to react with gastric cancer with high frequency, but not with normal gastric epithelium. Among normal digestive organs, only the colon and gall bladder were positive for MAb A3D4. The incidence of positivity in gastric cancer was 75% for intestinal-type adenocarcinoma (n = 28), 40% for solid-type adenocarcinoma (n = 5) and 33% for signet/scirrhous-type adenocarcinoma (n = 15). Interestingly, adenoma and intestinal metaplasia (IM) with chronic gastritis or peptic ulcer were negative for MAb A3D4, whereas 8 out of 13 cases (62%) of IM with gastric cancer was positive. Western-blot analysis using the lysate from normal colon tissues revealed a high-molecular-weight (> 300-kDa) smear-like band. Immunohistochemical analysis indicated that the reactivity of MAb A3D4 was clearly increased when tissue sections were pre-treated with periodic acid or O-glycanase, while it was decreased by pre-treatment with trypsin or protease V8. There was no reactivity with the synthetic peptide encompassing the tandem-repeat sequence of MUC2 or MUC3. These data suggest that MAb A3D4 detects a novel gastric-cancer-associated mucin antigen whose epitope may be peptide in nature.     

Human low-molecular-weight salivary mucin expresses the sialyl lewisx determinant and has L-selectin ligand activity

Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.     

Suppression of sigma spindle electroencephalographic activity as a measure of transient arousal after spontaneous and occlusion-evoked sighs and startles

Gastric M1 mucin and the MUC5AC gene show a similar oncofetal expression in the colon. Our aim was to determine whether M1 mucin is the product of the MUC5AC gene. A recombinant baculovirus encoding the C-terminal portion of the MUC5AC gene as a fusion protein was isolated and the immunoreactivity of the recombinant mucin (rM) toward M1 antibodies studied. Chicken antibodies also were raised against purified rM. Besides its reactivity with L56/C, a serum recognizing the bacterially expressed MUC5AC gene product, rM was endowed with M1 immunoreactivity: (i) rM-expressing cells were stained specifically with anti-M1 serum and with the monoclonal antibody (MAb) 21M1, defining the M1-f epitope; (ii) both L56/C and anti-M1 antibodies recognized the same bands in immunoblots of rM-containing cell extracts; (iii) the 21M1 antibody reacted with rM in an immunoradiometric assay. Among the 7 M1 epitopes, M1-f was the only one encoded by the 3' portion of the MUC5AC gene. It was the only epitope detected in a native mucin M1-derived 170 kDa bromelain proteolytic fragment. Furthermore, the staining patterns of human tissues obtained with either anti-rM chicken antibodies or anti-M1 antibodies were identical. We conclude that M1 immunoreactivity is encoded at least in part by the MUC5AC gene.     

Delivery of MUC1 mucin peptide by Poly(d,l-lactic-co-glycolic acid) microspheres induces type 1 T helper immune responses

Synthetic peptides corresponding to the variable tandem repeat domain of the cancer-associated antigen MUC1 mucin are candidates for cancer vaccines. In our investigation mice were immunized via subcutaneous injection with poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres containing a MUC1 mucin peptide. It was hypothesized that microencapsulation of the MUC1 mucin peptide would prime for antigen-specific Th1 responses while avoiding the need for traditional adjuvants and carrier proteins. Furthermore, an immunomodulator, monophosphoryl lipid A (MPLA), was incorporated into the peptide-loaded PLGA microspheres based on its ability to enhance Th1 responses. The results revealed T cell specific immune responses. The cytokine secretion profiles of the T cells consisted of high levels of interferon-gamma with undetectable levels of interleukin-4 and interleukin-10. Moreover, incorporation of MPLA in the MUC1 peptide-loaded PLGA microspheres resulted in an increase in interferon-gamma production. The antibody response was negative for IgM and IgG in the absence of MPLA; however, in the presence of MPLA antibody production was negative for IgM with a minimal IgG response consisting of IgG2a, IgG2b, and IgG3. Based on the antibody and cytokine profiles, it was concluded that MUC1 mucin peptide-loaded PLGA microspheres are capable of eliciting specific Th1 responses, which may be enhanced through the use of MPLA.     

Developmental mucin gene expression in the human respiratory tract

The epithelial surface of the respiratory tract is coated with a protective film of mucus secreted by epithelial goblet and submucosal gland cells. Histology of the airway mucosa and composition of secretions during the second trimester of fetal life are known to differ from the normal adult in that these secretions show similarities with those of hypersecretory disorders. To provide information regarding cell-specific expression of mucin genes and their relation to developmental patterns of epithelial cytodifferentiation, we studied the expression of eight different mucin genes (MUC1-MUC4, MUC5AC, MUC5B, MUC6, MUC7) in human embryonic and fetal respiratory tract using in situ hybridization. These investigations demonstrated that MUC4 is the earliest gene expressed in the foregut at 6.5 wk, followed by MUC1 and MUC2 from 9. 5 wk of gestation in trachea, bronchi, epithelial tubules, and terminal sacs before epithelial cytodifferentiation. In contrast, MUC5AC, MUC5B, and MUC7 are expressed at later gestational ages concomitant with epithelial cytodifferentiation. During this developmental stage, MUC1 and MUC4 mRNAs are located in goblet and ciliated cells, whereas MUC2 mRNAs are located in basal and goblet cells. MUC5AC expression is confined to goblet cells. In the submucosal glands, MUC2 mRNAs are located in both mucous and serous cells, whereas MUC5B and MUC7 mRNAs are expressed in mucous and in serous cells, respectively. These data suggest distinct developmental roles for MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7 in the elongation, branching, and epithelial cytodifferentiation of the respiratory tract during ontogenesis. Distinct patterns of mucin gene expression are also likely to play an important role in regulating appropriate epithelial cell proliferation and cytodifferentiation in adult airway mucosa as it is indicated by aberrant expression in hypersecretory disorders.     

Tissue antigens in the saliva of rats. Demonstration of salivary gland specific and nonspecific antigens

Antisera were raised in rabbits against the half-saturated ammonium sulfate soluble fraction of rat saliva. With these antisera, at least four antigens were demonstrated by immunodiffusion in the saliva and in a saline extract of the parotid gland; three of these antigens were also found in a saline extract of the submaxillary gland. Two of the four antigens were salivary gland specific, the other two being shared by the liver,pancreas and testis. The immunofluorescent pattern indicates that these antigens originate from the acinar epithelium of the salivary glands and probably constitute products of secretion and/or cellular turnover.     

Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps

Altered mucin glycosylation and the de novo appearance of gastric mucin antigens have been described in colonic adenomas. The purpose of our study was to determine if expression of the gastric mucin genes MUC5AC and MUC6 occurs in colorectal adenomas and whether this correlates with histopathologic criteria of malignant potential. Immunohistochemical staining using antibodies against MUC5AC and MUC6 tandem repeat synthetic peptides was performed on specimens of normal colon mucosa (n = 26), hyperplastic polyps (n = 9) and adenomatous polyps (n = 111). Mucin mRNA levels were determined using RNase protection assays using riboprobes corresponding to unique non-repetitive sequences. MUC5AC and MUC6 staining were rarely detected and of low intensity in normal colon and hyperplastic polyps. The number of immunoreactive polyps and intensity of MUC5AC and MUC6 staining were greatest in larger adenomas of moderate villous histology and dysplasia. MUC5AC and MUC6 staining tended to decrease in highly villous polyps with severe dysplasia. Increased MUC5AC mRNA levels were found in 26/45 of adenomas tested compared with 0/9 normal colon specimens. MUC6 mRNA levels were found in 20/45 of adenomas compared with 1/9 normal colon specimens. MUC5AC and MUC6 mRNA were present more frequently and at higher levels in polyps with intermediate stages of size, villous histology and dysplasia. We conclude that aberrant expression of MUC5AC and MUC6 mucin genes is likely responsible for an expanded repertoire of mucin antigen expression in colorectal neoplasia.     

Control of mucin molecular forms expression by salivary protease: differences with caries

1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion. 2. The protease exhibited optimum activity at pH 7.0-7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, alpha 1antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases. 3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects. 4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.     

Genetic salivary protein polymorphism in Mexican population

Genetic polymorphism is the major contributor that affects human salivary composition. In order to determine the molecular phenotypes in saliva, it is important to know the distribution of proteins with specific functions which allows the clinical diagnosis of specific diseases. Unstimulated human whole saliva samples from 120 subjects were subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE). The phenotype distribution of several molecules including MG1, MG2, alpha-Amylase, PRP-I and cystatins were similar. Qualitative and quantitative characteristics were specific in each subject.     

The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.     

Submandibular salivary proteases: lack of a role in anti-HIV activity

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.     

Structure, function and gene expression of epithelial mucins

In this review the main characteristics, i.e., structure, function and gene expression, of the different mucins are discussed. Mucin-type molecules consist of a core protein moiety (apomucin) where a number of carbohydrate chains are attached to serines and threonines by glycosidic bonds. O-linked carbohydrates form up to 80% of the molecule and the length of the glucidic side chains varies from one to more than 20 residues. At least eight mucin-like genes have been isolated so far, and the main characteristic is the presence of a central domain composed of a variable number of "tandem repeats". The sequence homology of the central domain among the different members of the mucin-type family is limited, indicating that this internal domain is unique for each mucin. Thanks to the integrated results of genetic, immunological and biochemical studies, it is now possible to identify eight apomucin genes, namely MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6 and MUC7. MUC1 is the best characterized mucin and it is expressed on the apical surface of most polarized epithelial cells. The MUC1 gene has been cloned and sequenced. The MUC2 gene encodes a typical secretory gel-forming mucin which represents the predominant form in human intestinal and colon tissues. Another intestinal mucin is MUC3. The MUC4, MUC5AC and MUC5B genes have been isolated from a bronchial tissue cDNA library. The MUC4 and MUC5AC genes are mainly expressed in the respiratory tract, in gastric and reproductive mucosa, while MUC5B is highly detectable only in the bronchial glands. The MUC6 gene is expressed by gastric tissue and, recently, MUC7 has been cloned and sequenced using a salivary cDNA library.     

The anti-MUC1 monoclonal antibody BCP8 can be used to isolate and identify putative major histocompatibility complex class I associated amino acid sequences

MHC class I molecules were isolated from the MUC1-positive human breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibodies (MAb) W6/32. Acid-eluted peptides from the class I molecules were separated twice by high-performance liquid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA. A peak with strong and specific BCP8 reactivity was found in fractions eluting at 16.5-17.5 min. The protocol used for the MUC1+ pancreatic adenocarcinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity purifications of class I molecules using MAb W6/32, followed by affinity purification of HLA.A2 molecules by the HLA.A2.1-specific MAb, MA2.1, and high-performance liquid chromatography fractionation of the acid-eluted material. A single peak with MAb BCP8 reactivity was noted at 18-19 min. The protocol for the MUC1+ breast adenocarcinoma cell line SKBr-3 (HLA.A11,B40), which used A11- and B40-specific MAbs, also resulted in the detection of BCP8-specific peaks at approximately 18-19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MUC1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to cause strong class I up-regulation in T2 cells as well as to serve as an epitope for CTL generated in a primary in vitro immune response. These studies suggest that MUC1-derived peptides are processed and presented in the context of MHC class I molecules on the surface of tumor cells and support the use of MAb BCP8 to further define MHC class I associated MUC1 motifs.     

Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by a N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus

Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.     

Augmenting the immunogenicity of synthetic MUC1 peptide vaccines in mice

Human mucin MUC1 is abundantly expressed in some cancers of epithelia] origin and is largely restricted to the apical surface of secretory cells in normal tissues. It is, therefore, a potential target for cancer immunotherapy. In preparation for clinical trials, vaccines containing synthetic MUC1 peptides of different lengths and sequences mixed with various adjuvants or covalently attached, using different linker methods, to protein carrier keyhole limpet hemocyanin (KLH) were studied in mice. MUC1 peptides (containing 30 amino acids), plus adjuvants QS-21 or Bacillus Calmette-Guerin, were incapable of inducing antibody. However, MUC1 peptides conjugated to KLH (MUCl-KLH), plus QS-21, induced high titer antibody against the immunizing peptides and against MUC1-expressing tumor cells. Although T-cell responses, including delayed-type hypersensitivity, lymphocyte proliferation, and CTL, were not observed in mice immunized with these vaccines, significant protection from MUC1-expressing tumor cell challenge in mice immunized with MUC1-KLH was observed. Based on these studies, a vaccine containing MUC1-KLH conjugate prepared with m-maleimidobenzoyl-N-hydroxysuccinimide ester linker, plus QS-21, has been constructed for testing in clinical trials.