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

Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
FEBS Journal (Impact Factor: 4). 07/2005; 272(11):2901-11. DOI: 10.1111/j.1742-4658.2005.04711.x
Source: PubMed


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.

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Available from: Simon Sherman, Nov 24, 2014
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    • "A comparison of the molecular structure and size of MUC1 and MUC16 (Fig. 1B) demonstrates that, of the two mucins, the ectodomain of MUC16 is about 20 times larger than that of MUC1 and its ectodomain includes a number of sea urchin sperm protein, enterokinase and agrin (SEA) modules, whereas MUC1 has one SEA module [7]. These modules are found in many membrane-associated proteins that are released from the cell surface [8]. "
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    ABSTRACT: Membrane-anchored mucins are present in the apical surface glycocalyx of mucosal epithelial cells, each mucosal epithelium having at least two of the mucins. The mucins have been ascribed barrier functions, but direct comparisons of their functions within the same epithelium have not been done. In an epithelial cell line that expresses the membrane-anchored mucins, MUC1 and MUC16, the mucins were independently and stably knocked down using shRNA. Barrier functions tested included dye penetrance, bacterial adherence and invasion, transepithelial resistance, tight junction formation, and apical surface size. Knockdown of MUC16 decreased all barrier functions tested, causing increased dye penetrance and bacterial invasion, decreased transepithelial resistance, surprisingly, disruption of tight junctions, and greater apical surface cell area. Knockdown of MUC1 did not decrease barrier function, in fact, barrier to dye penetrance and bacterial invasion increased significantly. These data suggest that barrier functions of membrane-anchored mucins vary in the context of other membrane mucins, and MUC16 provides a major barrier when present.
    PLoS ONE 06/2014; 9(6):e100393. DOI:10.1371/journal.pone.0100393 · 3.23 Impact Factor
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    • "If the molecule of 105 kDa was the carboxylterminus of chicken SPACR, it suggests that the molecule of 105 kDa and SPACR are from a single gene. The sea urchin, enterokinase, agrin (SEA) module is an extracellular domain present in a number of highly O-glycosylated membrane proteins, where it serves as a proteolytic cleavage site (Bork and Patthy 1995; Wreschner et al. 2002; Palmai-Pallag et al. 2005). SPACR, a heavily O-glycosylated protein, contains two SEA modules corresponding to amino acids 231–348 and 728–853 (Zako et al. 2002). "
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    ABSTRACT: The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.
    Journal of Neurochemistry 06/2008; 106(3):1117-24. DOI:10.1111/j.1471-4159.2008.05468.x · 4.28 Impact Factor
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    • "Processing of MUC1 proteins can result in both secreted and membrane-tethered variants, as demonstrated in Figure 1. The manner by which MUC1 undergoes cleavage has recently been described (Levitin et al. 2005; Palmai-Pallag et al. 2005; Macao et al. 2006), in which an enterokinase and agrin domain found in sea urchin sperm protein (SEA domain) generates the two polypeptides, MUC1-N and MUC1-C (Macao et al. 2006). The extra cellular fragment (MUC1-N) remains at the cell membrane by forming hetero-dimers with the transmembrane fragment (MUC1-C) (Palmai-Pallag et al. 2005). "
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    ABSTRACT: MUC1 is expressed on the apical surface of glandular epithelium. With functions including protection, adhesion and signaling, MUC1 has been implicated in prostate cancer. There are many splice variants, the best characterized of which are MUC1/1 and MUC1/2 which are determined by a SNP (rs4072037, 3506G>A). Blood DNA from the general population, BPH, sporadic and hereditary prostate cancer subjects were genotyped for the rs4072037 SNP. G allele frequencies were significantly reduced in hereditary prostate cancer (15%) compared to population, BPH or sporadic prostate cancer samples (27%, 39% and 26% respectively). In addition, the G allele was lost from 3 of 8 heterozygous sporadic prostate tumor samples compared to matched blood DNA. Bioinformatics analysis of MUC1 protein sequences provides insight into differences between the variants which may be functionally relevant. The literature indicates discrepancies between immuno-histochemical studies, possibly due to the variety of MUC1 epitopes targeting diverse regions of the molecule. The contradictory findings in cell lines highlight the problem associated with inadequate experimental systems. This is the first report of genetic differences in MUC1 between blood and prostatic cancer tissue. This finding is important as proof of principle, given that many association studies focus on blood DNA rather than on the tumor DNA. As yet, potential functional differences between splice variants has been paid little attention. Antibodies which discriminate between the variants and standardization of methods would help to clarify whether there is a role for MUC1 as a prognostic marker.
    Biomarker insights 02/2008; 3(3):303-315.
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