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, ␣ and ␤, 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 3 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.The MUC1 gene is highly expressed in a number of human epithelial malignancies, including breast, prostate, and colon carcinomas, as well as on the malignant plasma cells of multiple myeloma (1-9). As a well characterized tumor-associated protein, it has generated considerable interest as a tumor marker for disease prognosis (10 -14) as well as a target for tumor cell killing (15-18). Although alternative splicing can generate multiple MUC1 protein forms (19 -23), the most intensively studied MUC1 protein is a type I transmembrane protein comprised of a heavily glycosylated extracellular domain containing a tandem-repeat array, a transmembrane domain, and a cytoplasmic domain (Fig. 1, MUC1/TM) (24 -26). MUC1/TM is proteolytically cleaved soon after its synthesis, generating two subunits, ␣ and ␤, that specifically recognize each other and bind together by a strong noncovalent interaction (27).Cleavage occurs within the SEA module (28 -30), a highly conserved protein module so-called from its initial identification in a sperm protein, in enterokinase, and in agrin (31), that is found in a numb...

Disulfide bonds introduced during or shortly after protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent.

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 PATElike 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

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.Genes classified, for the most part, as MUC genes code for proteins that comprise mucin domains rich in proline, threonine, and serine residues (1). The heavily glycosylated mucin proteins derived from the MUC genes can be divided into those that are secreted from the cell (secreted mucins) and mucins that comprise a transmembrane domain that anchors them to the cell membrane (transmembrane mucins). The archetype membrane-tethered mucin, which was the one to be first characterized and sequenced, is derived from the MUC1 gene (2-5). It is a transmembrane protein that contains the serine-threonine-rich tandem-repeat region in its extracellular domain (Fig. 1A, MUC1/REP) and also comprises a 72 amino acid tail that can be tyrosine-phosphorylated (6, 7). The phosphorylated cytoplasmic domain subsequently interacts with second messenger proteins (6 -23), thereby relaying a signal to the nucleus that modifies gene expression.Challenging the classical definition of a mucin gene is the discovery of MUC1 mRNAs, which although transcribed from a mucin gene are devoid of a tandem-repeat array (24 -32). Alternative splicing generates these MUC1-derived mRNAs that no longer code for tandem-repeat array-containing mucin proteins. The prime example is the tandem-repeat array-deleted MUC1/Y isoform, which is expressed both as mRNA and protein (24 -26, 30, 31). The MUC1/Y protein is similar to MUC1/ REP in that it also contains the transmembrane and cytop...

background:The prostate and testis expression (PATE)-like family of proteins are expressed mainly in the male genital tract. They are localized in the sperm head and are homologous to SP-10, the acrosomal vesicle protein also named ACRV1. Our aim was to characterize the expression and functional role of three PATE-like proteins in the testis and ejaculated sperm. methods:The expression and localization of PATE-like proteins in human testis biopsies (n ¼ 95) and sperm cells were assessed by RT-PCR, immunohistochemistry and immunofluorescence staining (at least 600 sperm cells per specimen). The function of the PATE protein was tested by the hemizona assay and hamster egg penetration test (HEPT).results: PATE and PATE-M genes and proteins were present almost exclusively in germ cells in the testis: immunoflourescence showed that the percentage of germ cells positive for PATE, PATE-M and PATE-B was 85, 50 and 2%, respectively. PATE and PATE-M proteins were localized in the equatorial segment of the sperm head, while PATE-B protein was localized in the post-acrosomal region. A polyclonal antibody (Ab, at 1:50 and 1:200 dilutions) against the PATE protein did not inhibit sperm -zona binding in the hemizona assay (hemizona index of 89.6 + 10 and 87 + 36%, respectively). However, there was inhibition of sperm -oolemma fusion and penetration in the HEPT (penetration index: without Ab 7

Low levels of oxygen (hypoxia) occurs in many (patho)physiological situations. Adaptation to hypoxia is in part mediated by proteins expressed in the extracellular space that mature in the endoplasmic reticulum (ER) prior to traversing the secretory pathway. The majority of such ER cargo proteins require disulfide bonds for structural stability. Disulfide bonds are formed co- and posttranslationally in a redox relay that requires a terminal electron acceptor such as oxygen. We have previously demonstrated that some ER cargo proteins such as low-density lipoprotein receptor (LDLR) and influenza hemagglutinin (Flu-HA) are unable to complete disulfide bond formation in the absence of oxygen, limiting their ability to pass ER quality control and their ultimate expression. Here, using radioactive pulse-chase immunoprecipitation analysis, we demonstrate that hypoxia-induced ER cargo proteins such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A) complete disulfide bond formation and mature with similar kinetics under hypoxia and normoxia. A global in silico analysis of ER cargo revealed that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other ER cargo proteins. These data demonstrate the existence of alternative electron acceptors to oxygen for disulfide bond formation in cellulo . However, the ability of different proteins to utilize an oxygen-independent pathway for disulfide bond formation varies widely, contributing to differential gene expression in hypoxia. The superior ability of hypoxia-induced proteins such as VEGF-A and CA9 to mature in hypoxia may be conferred by a simpler disulfide architecture.

Tumor hypoxia results in poor patient outcome due to treatment resistance as well as biological changes that stimulate angiogenesis, vasculogenesis, migration, invasion and immune suppression. These hypoxia-induced adverse biological changes are often mediated by membrane bound or secreted proteins through transcriptional and translational upregulation. Thus, understanding the regulation of how secreted proteins in hypoxia can therefore reveal novel therapeutic targets. Proteins that traverse through the secretory pathway form disulfide bonds in the endoplasmic reticulum (ER). Recent data from our lab have demonstrated that disulfide bond formation remains incomplete in ER cargo proteins like LDLR and Flu-HA in the absence of oxygen. To address whether hypoxia-induced proteins were likewise impaired, radioactive pulse chase assays were performed to measure disulfide bond formation and secretion capacity under both normoxic and hypoxic conditions. Here, we demonstrate that both hypoxia induced proteins carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) complete disulfide bond formation and are secreted with equal kinetics under hypoxia and normoxia. These proteins hence have a superior ability to be expressed in the absence of oxygen. Additionally, in a global in silico analysis of all proteins that traverse through the ER, we discovered that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other proteins. These traits may contribute to their superior ability to form correct disulfide bonds in hypoxia. These data show that the ability of proteins to form native disulfide bonds in hypoxia varies widely which can ultimately contribute to their expression in the extracellular space. Citation Format: Sandy Che-Eun Serena Lee, Fiana Levitin, Stephanie Hulme, Ryan Rumantir, Jenna Sykes, Marianne Koritzinsky. Protein secretion rates of VEGF and CA9 in normoxia and hypoxia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3559.

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