In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies.

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

A small secreted protein NICOL regulates lumicrine-mediated sperm maturation and male fertility.

The mammalian spermatozoa produced in the testis require functional maturation in the epididymis for their full competence. Epididymal sperm maturation is regulated by lumicrine signalling pathways in which testis-derived secreted signals relocate to the epididymis lumen and promote functional differentiation. However, the detailed mechanisms of lumicrine regulation are unclear. Herein, we demonstrate that a small secreted protein, NELL2-interacting cofactor for lumicrine signalling (NICOL), plays a crucial role in lumicrine signalling in mice. NICOL is expressed in male reproductive organs, including the testis, and forms a complex with the testis-secreted protein NELL2, which is transported transluminally from the testis to the epididymis. Males lacking Nicol are sterile due to impaired NELL2-mediated lumicrine signalling, leading to defective epididymal differentiation and deficient sperm maturation but can be restored by NICOL expression in testicular germ cells. Our results demonstrate how lumicrine signalling regulates epididymal function for successful sperm maturation and male fertility.

LY6S, a New IFN-Inducible Human Member of the Ly6a Subfamily Expressed by Spleen Cells and Associated with Inflammation and Viral Resistance.

Syntenic genomic loci on human chromosome 8 and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte Ag 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to in this article as the Ly6a subfamily gene cluster. Ly6a, also known as Stem Cell Ag-1 and T cell-activating protein, is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report in this article on LY6S, a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the IFN-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a nonclassical spleen cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1-expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously unannotated human IFN-stimulated gene, LY6S, which has a 1:8 ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a nonclassical cell lineage, and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses.

Endogenous RNA cleavages at the ribosomal SRL site likely reflect miRNA (miR) mediated translational suppression.

We previously suggested a mechanism whereby the RNA induced silencing complex (RISC) brings about a specific cleavage at the sarcin-ricin loop (SRL) of 28S ribosomal RNA thereby eliciting translational suppression. Here we experimentally show that endogenous cleavages take place at the SRL site, in both mammalian cells and in Caenorhabditis elegans. Furthermore we demonstrate that bulged and looped-out residues present in the imperfect miRNA-[mRNA target site] duplexes, are complementary to the SRL site. These results support, and are compatible with, our described mechanism whereby microRNAs mediate cleavage of the highly conserved 28S rRNA sarcin/ricin loop leading to translational suppression.

ERK and PI3K regulate different aspects of the epithelial to mesenchymal transition of mammary tumor cells induced by truncated MUC1.

Epithelial to mesenchymal transition (EMT) integrates changes to cell morphology and signaling pathways resulting from modifications to the cell's transcriptional response. Different combinations of stimuli ignite this process in the contexts of development or tumor progression. The human MUC1 gene encodes multiple alternatively spliced forms of a polymorphic oncoprotein that is aberrantly expressed in epithelial malignancies. MUC1 is endowed with various signaling modules and has the potential to mediate proliferative and morphological changes characteristic of the progression of epithelial tumors. The tyrosine-rich cytoplasmic domain and the heavily glycosylated extracellular domain both play a role in MUC1-mediated signal transduction. However, the attribution of function to specific domains of MUC1 is difficult due to the concomitant presence of multiple forms of the protein, which stem from alternative splicing and proteolytic cleavage. Here we show that DA3 mouse mammary tumor cells stably transfected with a truncated genomic fragment of human MUC1 undergo EMT. In their EMT, these cells demonstrate altered [i] morphology, [ii] signaling pathways and [iii] expression of epithelial and mesenchymal markers. Similarly to well characterized human breast cancer cell lines, cells transfected with truncated MUC1 show an ERK-dependent increased spreading on fibronectin, and a PI3K-dependent enhancement of their proliferative rate.

The MUC1 oncoprotein as a functional target: immunotoxin binding to alpha/beta junction mediates cell killing.

MUC1, a heavily glycosylated mucin, has generated considerable interest as a target for tumor killing because of its overexpression in malignancies. Full-length MUC1 (MUC1/TM) is proteolytically cleaved after synthesis generating alpha and beta subunits, which specifically bind in a noncovalent interaction. Although the beta chain remains on the cell surface, the alpha chain binds in an on-and-off interaction. Most anti-MUC1 antibodies (Abs) described to date recognize epitopes within the highly immunogenic alpha-chain tandem repeat. Because the alpha-chain is shed, such Abs are sequestered and fail to reach MUC1-expressing cells. Immunizing with cDNA encoding MUC1/TM and the spliced MUC1/X isoform from which the tandem repeat has been deleted yielded antibodies to the MUC1 alpha/beta junction. Pseudomonas toxin PE38 linked to polyclonal anti-MUC1 alpha/beta junction Abs both bound and killed MUC1-positive malignant cells. Monoclonal DMC209 binds the MUC1 alpha/beta junction in both MUC1/X and MUC1/TM. When injected into SCID mice xenotransplanted with human breast cancer MDA-MB-231, monoclonal DMC209 showed significant in vivo tumor-suppressive activity. The MUC1/X alpha/beta junction presents a biologically-significant target in MUC1-expressing malignancies because (i) antibodies directed against cell-bound alpha/beta junction epitopes reach the intended cellular target, (ii) antibodies to junction epitope are internalized into cells, (iii) anti alpha/beta junction antibodies can effectively kill high MUC1-expressing cancer cells as antibody-toxin conjugates and (iv) antibodies targeting the MUC1 cell-bound alpha/beta junction results in tumor suppression in vivo. Our results indicate that cell-bound MUC1 alpha/beta junction, unlike shed alpha chain, represents a highly effective moiety for targeting and killing MUC1-expressing malignancies.

Targeting cell-bound MUC1 on myelomonocytic, monocytic leukemias and phenotypically defined leukemic stem cells with anti-SEA module antibodies.

Cell surface molecules aberrantly expressed or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1 is a polymorphic glycoprotein, the cleavage of which yields two unequal chains: a large extracellular α subunit containing a tandem repeat array bound in a strong noncovalent interaction to a smaller ß subunit containing the transmembrane and cytoplasmic domains. Because the α-chain can be released from the cell-bound domains of MUC1, agents directed against the α-chain will not effectively target MUC1+ cells. The MUC1 SEA (a highly conserved protein module so called from its initial identification in a sea urchin sperm protein, in enterokinase, and in agrin) domain formed by the binding of the α and ß chains  represents a stable structure fixed to the cell surface at all times. DMB-5F3, a partially humanized murine anti-MUC1 SEA domain monoclonal antibody, was used to examine MUC1 expression in acute myeloid leukemia (AML) and was found to bind acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5) cell lines. We also examined monocytic neoplasms freshly obtained from patients including chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, which were found to uniformly express MUC1. CD34+/lin-/CD38- or CD38+ presumed leukemic stem cell populations from CD34+ AML and CD34-CD38- or CD38+ populations from CD34- AML were also found to express MUC1, although at low percentages. Based on these studies, we generated an anti-MUC1 immunotoxin to directly gauge the cytotoxic efficacy of targeting AML-bound MUC1. Using single-chain DMB-5F3 fused to recombinant gelonin toxin, the degree of AML cytotoxicity was found to correlate with MUC1 expression. Our data support the use of an anti-MUC1 SEA module-drug conjugates to selectively target and inhibit MUC1-expressing myelomonocytic leukemic cells.

MUC1/X protein immunization enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target malignant cells.

MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most antibodies against MUC1 recognize epitopes within the highly immunogenic alpha chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 alpha chain is shed into the peripheral circulation, sequesters circulating antitandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recognizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 alpha subunit binding the membrane-tethered beta subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding full-length MUC1 (MUC1/TM) followed by boosting with the alternatively spliced MUC1/X isoform from which the tandem repeat array has been deleted, we generated monoclonal antibodies, designated DMC209, which specifically bind the MUC1 alpha/beta junction. DMC209 is exquisitely unique for this site; amino acid mutations, which abrogate MUC1 cleavage, also abrogate DMC209 binding. Additionally, DMC209 specifically binds the MUC1 alpha/beta junction on full-length MUC1/TM expressed by breast and ovarian cancer cell lines and on freshly obtained, unmanipulated MUC1-positive malignant plasma cells of multiple myeloma. DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly and as an immunotoxin conjugate. Moreover, the novel immunization procedure used in generating DMC209 can be used to generate additional anti-MUC1 alpha/beta junction antibodies, which may, analogously to Herceptin, have cytotoxic activity. Lastly, sequential immunization with MUC1/TM cDNA acting as a nonspecific adjuvant followed by protein of interest may prove to be a generalizable method to yield high-titer specific antibodies.

MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERalpha) in regulation of the MUC1 gene expression.

BACKGROUND: The MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter. RESULTS: Using TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERalpha, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed. CONCLUSION: Analysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERalpha in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy.

A unique mucin immunoenhancing peptide with antitumor properties.

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.

MUC1 immunotherapy against a metastatic mammary adenocarcinoma model: Importance of IFN-gamma.

Immunotherapy using mucin 1 (MUC1) linked to oxidised mannan (MFP) was investigated in an aggressive MUC1+ metastatic tumour, DA3-MUC1 because, unlike many MUC1+ tumour models, DA3-MUC1 is not spontaneously rejected in mice making it an alternative model for immunotherapy studies. Further, DA3-MUC1 cells are resistant to lysis by anti-MUC1 cytotoxic T cells (CTLs). The inability of DA3-MUC1 tumours to be rejected in naïve mice as well as vaccination to MUC1 was attributed to a deficiency of expression of MHC class I molecules on the tumour cell surface. In vitro and in vivo analysis of subcutaneous tumours and lung metastases demonstrated that DA3-MUC1 tumour cells have a low expression (< 6%) of MHC class I which can be upregulated (> 90%) following culturing with IFN-γ. Results from flow cytometry analysis and immunoperoxidase staining indicated that the in vitro up-regulation of MHC class I could be maintained for up to seven days in vivo, without affecting the expression levels of MUC1 antigen. Interestingly, MUC1-specific CTL that lyse DA3-MUC1 targets in vitro were induced in MFP immunised mice but failed to protect mice from a DA3-MUC1 tumour challenge. These results highlight the importance of MHC class I molecules in the induction of anti-tumour immunity and the MFP immune response.

MUC1-ARF-A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA.

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described.

Generation of ligand-receptor alliances by "SEA" module-mediated cleavage of membrane-associated mucin proteins.

A mechanism is described whereby one and the same gene can encode both a receptor protein as well as its specific ligand. Generation of this receptor-ligand partnership is effected by proteolytic cleavage within a specific module located in a membrane resident protein. It is postulated here that the "SEA" module, found in a number of heavily O-linked glycosylated membrane-associated proteins, serves as a site for proteolytic cleavage. The subunits generated by proteolytic cleavage of the SEA module reassociate, and can subsequently elicit a signaling cascade. We hypothesize that all membrane resident proteins containing such a "SEA" module will undergo cleavage, thereby generating a receptor-ligand alliance. This requires that the protein subunits resulting from the proteolytic cleavage reassociate with each other in a highly specific fashion. The same SEA module that serves as the site for proteolytic cleavage, probably also contains the binding sites for reassociation of the resultant two subunits. More than one type of module can function as a site for proteolytic cleavage; this can occur not only in one-pass membrane proteins but also in 7-transmembrane proteins and other membrane-associated proteins. The proposal presented here is likely to have significant practical consequences. It could well lead to the rational design and identification of molecules that, by binding to one of the cleaved partners, will act either as agonists or antagonists, alter signal transduction and, hence, cellular behavior.

Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube.

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.

Antibody targeting of cell-bound MUC1 SEA domain kills tumor cells.

The cell-surface glycoprotein MUC1 is a particularly appealing target for antibody targeting, being selectively overexpressed in many types of cancers and a high proportion of cancer stem-like cells. However the occurrence of MUC1 cleavage, which leads to the release of the extracellular α subunit into the circulation where it can sequester many anti-MUC1 antibodies, renders the target problematic to some degree. To address this issue, we generated a set of unique MUC1 monoclonal antibodies that target a region termed the SEA domain that remains tethered to the cell surface after MUC1 cleavage. In breast cancer cell populations, these antibodies bound the cancer cells with high picomolar affinity. Starting with a partially humanized antibody, DMB5F3, we created a recombinant chimeric antibody that bound a panel of MUC1+ cancer cells with higher affinities relative to cetuximab (anti-EGFR1) or tratuzumab (anti-erbB2) control antibodies. DMB5F3 internalization from the cell surface occurred in an efficient temperature-dependent manner. Linkage to toxin rendered these DMB5F3 antibodies to be cytotoxic against MUC1+ cancer cells at low picomolar concentrations. Our findings show that high-affinity antibodies to cell-bound MUC1 SEA domain exert specific cytotoxicity against cancer cells, and they point to the SEA domain as a potential immunogen to generate MUC1 vaccines.

Similarities between Argonautes and the alpha-sarcin-like ribotoxins: Implications for microRNA action.

We report structural, functional, and biochemical similarities between Argonautes, the effector proteins of RNA-induced silencing complexes (RISCs), and alpha-sarcin-like ribotoxins. At the structural level, regions of similarity in the amino acid sequence are located in protein loops both in the ribotoxins and in the Argonautes. In ribotoxins, these protein loops confer specificity for a highly conserved segment of ribosomal RNA, the Sarcin-Ricin-Loop (SRL) that undergoes cleavage by the ribotoxin ribonuclease. This leads to suppression of translation. In addition to the structural similarity with ribotoxins, the Argonaute proteins (Ago) show both functional and biochemical parallels. Like the ribotoxins, the Agos exhibit ribonuclease activity and like the ribotoxins, translational suppression mediated by miRISC-resident Ago is accompanied by intact polysomes. Furthermore, in both translationally suppressed systems, the puromycin reaction, reflecting correct translocation and peptidyl-transferase activities, is unharmed. These findings support a mechanism for Ago-miRISCs whereby regulated cleavage of ribosomal RNA leads to translational suppression.

Protein multifunctionality: principles and mechanisms.

In the review, the nature of protein multifunctionality is analyzed. In the first part of the review the principles of structural/functional organization of protein are discussed. In the second part, the main mechanisms involved in development of multiple functions on a single gene product(s) are analyzed. The last part represents a number of examples showing that multifunctionality is a basic feature of biologically active proteins.

PATE gene clusters code for multiple, secreted TFP/Ly-6/uPAR proteins that are expressed in reproductive and neuron-rich tissues and possess neuromodulatory activity.

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATE-like genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region of the sperm acrosome and at high levels on malignant prostatic epithelial cells. The syntenic mouse Pate-like locus encompasses 14 active genes coding for secreted proteins, which are all, except for Pate-P and Pate-Q, expressed primarily in prostate and/or testis. Pate-P and Pate-Q are expressed solely in placental tissue. Castration up-regulates prostate expression of mouse Pate-B and Pate-E, whereas testosterone ablates this induced expression. The sequence similarity between TFP/Ly-6/uPAR proteins that modulate activity of nicotinic acetylcholine receptors and the PATE (Pate)-like proteins stimulated us to see whether these proteins possess analogous activity. Pharmacological studies showed significant modulation of the nicotinic acetylcholines by the PATE-B, Pate-C, and Pate-P proteins. In concert with these findings, certain PATE (Pate)-like genes were extensively expressed in neuron-rich tissues. Taken together, our findings indicate that in addition to participation of the PATE (Pate)-like genes in functions related to fertility and reproduction, some of them likely act as important modulators of neural transmission.

The MUC1 SEA module is a self-cleaving domain.

MUC1, a glycoprotein overexpressed by a variety of human adenocarcinomas, is a type I transmembrane protein (MUC1/TM) that soon after its synthesis undergoes proteolytic cleavage in its extracellular domain. This cleavage generates two subunits, alpha and beta, that specifically recognize each other and bind together in a strong noncovalent interaction. Proteolysis occurs within the SEA module, a 120-amino acid domain that is highly conserved in a number of heavily glycosylated mucin-like proteins. Post-translational cleavage of the SEA module occurs at a site similar to that in MUC1 in the glycoproteins IgHepta and MUC3. However, as in the case of other proteins containing the cleaved SEA module, the mechanism of MUC1 proteolysis has not been elucidated. Alternative splicing generates two transmembrane MUC1 isoforms, designated MUC1/Y and MUC1/X. We demonstrated here that MUC1/X, whose extracellular domain is comprised solely of the SEA module in addition to 30 MUC1 N-terminal amino acids, undergoes proteolytic cleavage at the same site as the MUC1/TM protein. In contrast, the MUC1/Y isoform, composed of an N-terminally truncated SEA module, is not cleaved. Cysteine or threonine mutations of the MUC1/X serine residue (Ser-63) immediately C-terminal to the cleavage site generated cleaved proteins, whereas mutation of the Ser-63 residue of MUC1/X to any other of 17 amino acids did not result in cleavage. In vitro incubation of highly purified precursor MUC1/X protein resulted in self-cleavage. Furthermore, addition of hydroxylamine, a strong nucleophile, markedly enhanced cleavage. Both these features are signature characteristics of self-cleaving proteins, and we concluded that MUC1 undergoes autoproteolysis mediated by an N --> O-acyl rearrangement at the cleavage site followed by hydrolytic resolution of the unstable ester and concomitant cleavage. It is likely that all cleaved SEA module-containing proteins follow a similar route.

A novel protein derived from the MUC1 gene by alternative splicing and frameshifting.

Genes that have been designated the name "MUC" code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.