Sea Urchin Sperm Protein Research Articles

Unraveling mucin domains in cancer and metastasis: when protectors become predators.

A dynamic mucosal layer shields the epithelial cells lining the body cavities and is made up of high molecular weight, heavily glycosylated, multidomain proteins called mucins. Mucins, broadly grouped into transmembrane and secreted mucins, are the first responders to any mechanical or chemical insult to the epithelia and help maintain tissue homeostasis. However, their intrinsic properties to protect and repair the epithelia are exploited during oncogenic processes, where mucins are metamorphosed to aid the tumor cells in their malignant journey. Diverse domains, like the variable number tandem repeats (VNTR), sea urchin sperm protein enterokinase and agrin (SEA), adhesion-associated domain (AMOP), nidogen-like domain (NIDO), epidermal growth factor-like domain (EGF), and von Willebrand factor type Ddomain (vWD) on mucins, including MUC1, MUC4, MUC5AC, MUC5B, and MUC16, have been shown to facilitate cell-to-cell and cell-to-matrix interactions, and cell-autonomous signaling to promote tumorigenesis and distant dissemination of tumor cells. Several obstacles have limited the study of mucins, including technical difficulties in working with these huge glycoproteins, the dearth of scientific tools, and lack of animal models; thus, the tissue-dependent and domain-specific roles of mucins during mucosal protection, chronic inflammation, tumorigenesis, and hematological dissemination of malignant cells are still unclear. Future studies should try to integrate information on the rheological, molecular, and biological characteristics of mucins to comprehensively delineate their pathophysiological role and evaluate their suitability as targets in future diagnostic and therapeutic strategies.

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 β 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 andβchains 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.

Abstract 3427: Targeting MUC13 to overcome the survival mechanisms for improved response to chemotherapy

Abstract Background: Pancreatic cancer (PanCa) is a third leading cause of cancer-related deaths in the US due to late diagnosis and development of chemoresistance. Therefore, understanding molecular mechanisms that confer survival benefit to PanCa cells may offer new therapeutic strategies for PanCa treatment.Mucin, MUC13 aberrantly expressed in PanCa,promoting cancer growth and progression and these effects are abrogated by microRNA-145 (miR-145) restoration. Unlike other cancer types, PanCa is highly resistant to Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) that emerges as one of the most promising experimental cancer therapeutic drugs.Herein, we demonstrate the integration of novel approach to overcome chemoresistance and offer TRAIL-based therapeutic strategies. Methods: MUC13 expressing and null stable PanCa cells were generated to investigate the role of MUC13 in cell survival.miR-145 mimics were used to investigate the effect of MUC13 silencing in promoting survival and inhibiting apoptosis in presence of TRAIL using Western blotting, cell proliferation (MTT), Acridine orange staining and flow cytometry apoptosis assays(cell cycle, Annexin V/7AAD staining). Various distinct domain specific constructs of MUC13 were constructed such as the mucin (α), Beta (β) sea urchin sperm protein enterokinase arginine (SEA) domain and cytoplasmic (CD) domains and transfected into MUC13 null Panc-1 cells to identify the role of different domains in eliciting survival benefit to PanCa cells. Results: Results demonstrate that MUC13 expression blocks activation of caspase-8 and death receptor-mediated apoptosis in PanCa cells in response toTRAIL treatment as observed through Western blotting and flow cytometer. Inhibition of MUC13 using shRNA knockdown or miR-145 restoration resulted in the TRAIL-mediated increase in apoptotic cell death as evidenced by AnnexinV/ 7AAD and sub G0 population, as well as rendered PanCa cells sensitive to treatment with drugs, such as paclitaxel. Additionally, cells treated with TRAIL in combination with paclitaxel or Abraxane showed enhanced apoptosis on inhibition of MUC13 expression using miR-145 restoration. Further investigation showed that cytoplasmic domain of MUC13 (MUC13-CD) is indispensable for blockingcaspase-8 activation and PARP cleavage, indicating that the MUC13-CD blocks TRAIL-induced signaling upstream to Bid by inhibiting caspase-8 activation. Conclusion: These observations suggest that MUC13 contributes to the survival advantage in PanCa cells in response to treatment with drugs or death inducing ligands such as, Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) which can be strategically overcome by miR-145 replenishment. These findings indicate that MUC13 silencing sensitizes PanCa cells towards TRAIL therapy and counteracts chemoresistance mechanisms in PanCa that may lead to novel combination therapies for PanCa treatment. Citation Format: Saini Setua, Sheema Khan, Murali M. Yallapu, Stephen W. Behrman, Nadeem Zafar, Meena Jaggi, Subhash C. Chauhan. Targeting MUC13 to overcome the survival mechanisms for improved response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3427. doi:10.1158/1538-7445.AM2017-3427

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54.

SEA (sea urchin sperm protein, enterokinase, agrin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor-type protein phosphatase IA-2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA-2, and Notch receptors. By comparative sequence and structural analyses, we identified dystroglycan-like proteins with SEA domains in Capsaspora owczarzaki of the Filasterea group, one of the closest single-cell relatives of metazoans. We also detected novel and divergent SEA domains in a variety of cell surface proteins such as EpCAM, α/ε-sarcoglycan, PTPRR, collectrin/Tmem27, amnionless, CD34, KIAA0319, fibrocystin-like protein, and a number of cadherins. While these proteins are mostly from metazoans or their single cell relatives such as choanoflagellates and Filasterea, fibrocystin-like proteins with SEA domains were found in several other eukaryotic lineages including green algae, Alveolata, Euglenozoa, and Haptophyta, suggesting an ancient evolutionary origin. In addition, the intracellular protein Nucleoporin 54 (Nup54) acquired a divergent SEA domain in choanoflagellates and metazoans.

Characterization of a Gene Family Encoding SEA (Sea-urchin Sperm Protein, Enterokinase and Agrin)-Domain Proteins with Lectin-Like and Heme-Binding Properties from Schistosoma japonicum

BackgroundWe previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information.Methodology/Principal FindingsHere, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10−6 M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition.ConclusionsThe results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

A Mechanistic Study of the Spontaneous Hydrolysis of Glycylserine as the Simplest Model for Protein Self‐Cleavage

A common feature of several classes of intrinsically reactive proteins with diverse biological functions is that they undergo self-catalyzed reactions initiated by an N→O or N→S acyl shift of a peptide bond adjacent to a serine, threonine, or cysteine residue. In this study, we examine the N→O acyl shift initiated peptide-bond hydrolysis at the serine residue on a model compound, glycylserine (GlySer), by means of DFT and ab initio methods. In the most favorable rate-determining transition state, the serine COO(-) group acts as a general base to accept a proton from the attacking OH function, which results in oxyoxazolidine ring closure. The calculated activation energy (29.4 kcal mol(-1) ) is in excellent agreement with the experimental value, 29.4 kcal mol(-1) , determined by (1) H NMR measurements. A reaction mechanism for the entire process of GlySer dipeptide hydrolysis is also proposed. In the case of proteins, we found that when no other groups that may act as a general base are available, the N→O acyl shift mechanism might instead involve a water-assisted proton transfer from the attacking serine OH group to the amide oxygen. However, the calculated energy barrier for this process is relatively high (33.6 kcal mol(-1) ), thus indicating that in absence of catalytic factors the peptide bond adjacent to serine is no longer a weak point in the protein backbone. An analogous rearrangement involving the amide N-protonated form, rather than the principle zwitterion form of GlySer, was also considered as a model for the previously proposed mechanism of sea-urchin sperm protein, enterokinase, and agrin (SEA) domain autoproteolysis. The calculated activation energy (14.3 kcal mol(-1) ) is significantly lower than the experimental value reported for SEA (≈21 kcal mol(-1) ), but is still in better agreement as compared to earlier theoretical attempts.

Crystal Structures of Matriptase in Complex with Its Inhibitor Hepatocyte Growth Factor Activator Inhibitor-1

Matriptase, a type II trans-membrane serine protease of the S1 trypsin-like family, is expressed on the surface of nearly all normal human epithelium and found in biological fluid-like human milk. Matriptase overexpression has been implicated in tumor progression in certain epithelium-derived cancer cells. Matriptase is tightly regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). It has been demonstrated that the Kunitz domain I (KD1) but not Kunitz domain II (KD2) of HAI-1 is responsible for the inhibitory activity of HAI-1 against matriptase. To investigate the molecular basis of inhibition of matriptase by HAI-1, we solved several crystal structures of matriptase serine protease domain in complex with the fragments of HAI-1. Based on these structures, we found that the binding of KD1 was different from previously predicted binding mode. The P3 arginine residue occupies the S3 specificity pocket of matriptase, but not the S4 pocket as in the cases of hepatocyte growth factor activator·HAI-1 KD1 and matriptase·sunflower trypsin inhibitor-1 complexes. The long 60-loop of matriptase makes direct contact with HAI-1 but remains flexible even in the complexes, and its apex does not bind with KD1 tightly. The interactions between this unique 60-loop and KD1 may provide an opportunity to increase the specificity and inhibitory activity of KD1 for matriptase. Furthermore, comparison between KD1 and a homology model of HAI-1 KD2 rationalizes the structural basis of why KD1 but not KD2 is responsible for the inhibitory activity of HAI-1 against matriptase.

Unfolding dynamics of the mucin SEA domain probed by force spectroscopy suggest that it acts as a cell‐protective device

MUC1 and other membrane-associated mucins harbor long, up to 1 μm, extended highly glycosylated mucin domains and sea urchin sperm protein, enterokinase and agrin (SEA) domains situated on their extracellular parts. These mucins line luminal tracts and organs, and are anchored to the apical cell membrane by a transmembrane domain. The SEA domain is highly conserved and undergoes a molecular strain-dependent autocatalytic cleavage during folding in the endoplasmic reticulum, a process required for apical plasma membrane expression. To date, no specific function has been designated for the SEA domain. Here, we constructed a recombinant protein consisting of three SEA domains in tandem and used force spectroscopy to assess the dissociation force required to unfold individual, folded SEA domains. Force-distance curves revealed three peaks, each representing unfolding of a single SEA domain. Fitting the observed unfolding events to a worm-like chain model yielded an average contour length of 32 nm per SEA domain. Analysis of forces applied on the recombinant protein revealed an average unfolding force of 168 pN for each SEA domain at a loading rate of 25 nN·s(-1). Thus, the SEA domain may act as a breaking point that can dissociate before the plasma membrane is breached when mechanical forces are applied to cell surfaces.

The quest for the sea urchin egg receptor for sperm

This review discusses identification, isolation and characterization of proteins mediating species-selective sperm-to-egg adhesion during sea urchin fertilization. Bindin is the only sea urchin sperm protein known to mediate species-selective sperm attachment to eggs. Two completely different egg surface proteins, 350-kDa and EBR1, have affinity for bindin and each one meets all the criteria to be a species-selective sperm receptor. Experiments suggest that sperm bindin recognizes both the sulfated O-linked oligosaccharides on the egg 350-kDa glycoprotein, and also the repeated protein sequence modules of EBR1.

Neurobin/TMPRSS11c, a novel type II transmembrane serine protease that cleaves fibroblast growth factor-2 in vitro

TTSPs [type II TMPRSSs (transmembrane serine proteases)] are a growing family of trypsin-like enzymes with, in some cases, restricted tissue distribution. To investigate the expression of TTSPs in the nervous system, we performed a PCR-based screening approach with P10 (postnatal day 10) mouse spinal cord mRNA. We detected the expression of five known TTSPs and identified a novel TTSP, which we designated neurobin. Neurobin consists of 431 amino acids. In the extracellular part, neurobin contains a single SEA (sea-urchin sperm protein, enterokinase and agrin) domain and a C-terminal serine protease domain. RT-PCR (reverse transcription-PCR) analysis indicated the expression of neurobin in spinal cord and cerebellum. Histochemical analysis of brain sections revealed distinct staining of Purkinje neurons of the cerebellum. Transiently overexpressed neurobin was autocatalytically processed and inserted into the plasma membrane. Autocatalytic activation could be suppressed by mutating Ser(381) in the catalytic pocket to an alanine residue. The protease domain of neurobin, produced in Escherichia coli and refolded from inclusion bodies, cleaved chromogenic peptides with an arginine residue in position P(1). Serine protease inhibitors effectively suppressed the proteolytic activity of recombinant neurobin. Ca2+ or Na+ ions did not significantly modulate the catalytic activity of the protease. Recombinant neurobin processed 17-kDa FGF-2 (fibroblast growth factor-2) at several P(1) lysine and arginine positions to distinct fragments, in a heparin-inhibitable manner, but did not cleave FGF-7, laminin or fibronectin. These results indicate that neurobin is an authentic TTSP with trypsin-like activity and is able to process FGF-2 in vitro.

Contribution of the conservative cleavage motif to posttranslational processing of the carboxyl terminal domain of rodent Muc3

In previous works, we showed by transient expression studies in COS-1 cells that the C-terminal domain of rat intestinal membrane mucin (rMuc3) that was cloned in the pSecTag2 plasmid (named as p20) is posttranslationally cleaved twice. One location is between the glycine and the serine within a LS1KGS2IV1V2 motif, and the other is in the 49 kDa membrane-tethered fragment at an undefined site. The sea-urchin sperm protein, enterokinase and agrin module of rMuc3 is responsible for the cleavage and association of the cleaved fragments. The present study demonstrates how the conservative cleavage motif LS1KGS2IV1V2 contributes to posttranslational processing through mutagenesis of each residue in the LS(1)KGS2IV1V2 motif. Mutation of S2 to alanine (p20s2/a) completely prevented cleavage. While p20k/a (in this construct the K is replaced by A) and p20s1/a (in this construct the S1 is replaced by A) (6 and 3%) showed almost the same result as the wild-type p20 transfectant (4%), 79, 39, 22, 17, and 14% of the products from p20g/a (in this construct the G is replaced by A), p20i/a (in this construct the I is replaced by A), p20l/a (in this construct the L is replaced by A), p20v2/a (in this construct the V2 is replaced by A), and p20v1/a (in this construct the V1 is replaced by A) remained uncleaved. The cleaved N-terminal fragment of the p20s1/a transfectant was 26 kDa, but the N-terminal fragments from p20, p20g/a, p20l/a, p20k/a, p20i/a, p20v1/a, and p20v2/a were 30 kDa. The S1 residue was possibly O-glycosylated, which was supported by deglycosylation with O-cocktail (a mixture of glycosidases). The N-terminal fragment of p20s1/a transfected cells was present at high levels in the spent media. Thus, the S2, G, I, L, V2, and V1 residues within the conserved cleavage motif, LS1KGS2IV1V2, are important for cleavage and contribute to the structural formation and conformational stress of the small loop between the beta2 and the beta3 strands. The S1 residue is possibly O-glycosylated, and mutation of S1 residue to alanine does not affect the cleavage of the LS1KGS2IV1V2 motif, but it is important for the dissociation and further release of the cleaved N-terminal fragment from the cell surface.

Structure of the Mature Ectodomain of the Human Receptor-type Protein-tyrosine Phosphatase IA-2

IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30A resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo- and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions.

SEA Domain Autoproteolysis Accelerated by Conformational Strain: Mechanistic Aspects

A subclass of SEA (sea urchin sperm protein, enterokinase, and agrin) domain proteins undergoes autoproteolysis between glycine and serine in a conserved G−1S+1VVV motif to generate stable heterodimers. Autoproteolysis has been suggested to involve only the intramolecular catalytic action of the conserved serine hydroxyl in combination with conformational strain of the glycine–serine peptide bond. We conducted a number of experiments and simulations on the SEA domain from the MUC1 mucin to test this mechanism. Alanine-scanning mutagenesis of polar residues in the vicinity of the cleavage site demonstrates that only the nucleophile at position +1 is required for efficient proteolysis. Molecular modeling shows that an uncleaved trans peptide is incompatible with the native heterodimeric structure, resulting in disruption of secondary structure elements and distortion of the scissile peptide bond. Insertion of glycine residues (to obtain GnG−1S+1VVV motifs) appears to relieve strain, and autoproteolysis is 100 times slower in a 1G (n=1) mutant and not measurable in 2G and 4G mutants. Removal of the catalytic serine hydroxyl hampers cleavage considerably, but measurable autoproteolysis of this S1098A mutant still proceeds in the presence of strain alone. The uncleaved SEA precursor populates interconverting partially folded conformations, and autoproteolysis coincides with adoption of proper β-sheet secondary structure and completed folding. Molecular dynamics simulations of the precursor show that the serine hydroxyl and the preceding glycine carbonyl carbon can be in van der Waals contact at the same time as the scissile peptide bond becomes strained. These observations are all consistent with autoproteolysis accelerated by N→O acyl shift and conformational strain imposed upon protein folding in a reaction for which the free-energy barrier is decreased by substrate destabilization rather than by transition-state stabilization. The energetics of this coupled folding and autoproteolysis mechanism is accounted for in an accompanying article.

SEA Domain Autoproteolysis Accelerated by Conformational Strain: Energetic Aspects

A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G−1S+1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N→O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t½) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by ∼10 kcal mol−1 compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to ∼11 and ∼15 kcal mol−1, respectively, but ∼7 kcal mol−1 of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of ∼7 kcal mol−1 was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G−1A+1VVV motif) and the wild-type protein. The results imply that cleavage by N→O acyl shift alone would proceed with a t½ of ∼2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.

Serase-1B, a new splice variant of polyserase-1/TMPRSS9, activates urokinase-type plasminogen activator and the proteolytic activation is negatively regulated by glycosaminoglycans

Polyserase-1 (polyserine protease-1)/TMPRSS9 (transmembrane serine protease 9) is a type II transmembrane serine protease (TTSP) that possesses unique three tandem serine protease domains. However, the physiological function of each protease domain remains poorly understood. We discovered a new splice variant of polyserase-1, termed Serase-1B, which contains 34 extra amino acids consisting a SEA module (a domain found in sea urchin sperm protein, enterokinase and agrin) adjacent to the transmembrane domain and the first protease domain with a mucin-like box at the C-terminus. The tissue distribution of this enzyme by RT (reverse transcription)-PCR analysis revealed high expression in the liver, small intestine, pancreas, testis and peripheral blood CD14+ and CD8+ cells. To investigate the role of Serase-1B, a full-length form recombinant protein was produced. Interestingly, recombinant Serase-1B was partly secreted as a soluble inactive precursor and it was also activated by trypsin. This activated enzyme selectively cleaved synthetic peptides for trypsin and activated protein C, and it was inhibited by several natural serine protease inhibitors, such as aprotinin, alpha2-antiplasmin and plasminogen activator inhibitor 1. In addition, Serase-1B efficiently converted pro-uPA (urokinase-type plasminogen activator) into active uPA and this activation was strongly inhibited by these natural inhibitors. Furthermore, this activation was also negatively regulated by glycosaminoglycans. Our results indicate that Serase-1B is a novel member of TTSPs that might be involved in uPA/plasmin-mediated proteolysis and possibly implicated in biological events such as fibrinolysis and tumour progression.

The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane‐tethered mucins

The membrane-tethered mucins are cell surface-associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane-associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane-tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site-directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately re-create an accessible cleavage site.

Mouse DESC1 Is Located within a Cluster of Seven DESC1-like Genes and Encodes a Type II Transmembrane Serine Protease That Forms Serpin Inhibitory Complexes

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.

Initial characterization of an uromodulin-like 1 gene on human chromosome 21q22.3

We have isolated a novel gene, designated UMODL1, similar to uromodulin ( UMOD)/Tamm–Horsfall glycoprotein, on human chromosome 21q22.3. Uromodulin like-1 ( UMODL1) consists of 22 exons and spans approximately 80 kb in a direction from centromere to telomere. Two major transcripts produced by alternative splicing have been identified. These transcripts contain open reading frames of 4125 and 3741 bp encoding proteins of 1374 and 1246 amino acids, respectively. Expression of UMODL1 mRNA was detected only in 14 human tissues, e.g., kidney, testis, and fetal thymus at low level. Interestingly, two gene products (UMODL1L and UMODL1S) contain multiple domains including whey acidic protein, sea urchin sperm protein, enterokinase, and agrin, zona pellucida domain, and so on. Both proteins seemed to localize in cytoplasm, but UMODL1 is likely to be ubiquitinated and rapidly degraded in HEK293 cells. This gene may be a potent candidate for Down syndrome or bipolar affective disorder.

Evidence for a second peptide cleavage in the C-terminal domain of rodent intestinal mucin Muc3.

Rat intestinal mucin Muc3 (rMuc3), like its human homologue (MUC3) and several other membrane mucins, contains a C-terminally located SEA (sea urchin sperm protein, enterokinase and agrin) module, with an intrinsic proteolytic site sequence G downward arrow SIVV (where G downward arrow S is the glycine serine cleavage site). As shown previously [Wang, Khatri and Forstner (2002) Biochem. J. 366, 623-631], expression of the C-terminal domain of rMuc3 in COS-1 cells yields a V5 epitope-tagged N-terminal glycopeptide of 30 kDa and a Myc- and His epitope-tagged C-terminal glycopeptide of 49 kDa. The present study shows that the 49 kDa membrane-anchored fragment undergoes a further cleavage reaction which decreases its size to 30 kDa. Western blotting, pulse-chase metabolic incubations, immunoprecipitation and deglycosylation with N-glycosidase F were used to detect and identify the proteolytic products. Both the first and second cleavages are presumed to facilitate solubilization of Muc3 at the apical surface of enterocytes and/or enhance the potential for Muc3 to participate in ligand-receptor and signal transduction events for enterocyte function in vivo.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease.

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat (RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT (HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 (AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract-but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.