mua-3, a gene required for mechanical tissue integrity in Caenorhabditis elegans, encodes a novel transmembrane protein of epithelial attachment complexes

Normal locomotion of the nematode Caenorhabditis elegans requires transmission of contractile force through a series of mechanical linkages from the myofibrillar lattice of the body wall muscles, across an intervening extracellular matrix and epithelium (the hypodermis) to the cuticle. Mutations in mua-3 cause a separation of the hypodermis from the cuticle, suggesting this gene is required for maintaining hypodermal-cuticle attachment as the animal grows in size postembryonically. mua-3 encodes a predicted 3,767 amino acid protein with a large extracellular domain, a single transmembrane helix, and a smaller cytoplasmic domain. The extracellular domain contains four distinct protein modules: 5 low density lipoprotein type A, 52 epidermal growth factor, 1 von Willebrand factor A, and 2 sea urchin-enterokinase-agrin modules. MUA-3 localizes to the hypodermal hemidesmosomes and to other sites of mechanically robust transepithelial attachments, including the rectum, vulva, mechanosensory neurons, and excretory duct/pore. In addition, it is shown that MUA-3 colocalizes with cytoplasmic intermediate filaments (IFs) at these sites. Thus, MUA-3 appears to be a protein that links the IF cytoskeleton of nematode epithelia to the cuticle at sites of mechanical stress.     

M142.2 (cut-6), a novel Caenorhabditis elegans matrix gene important for dauer body shape

The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6.     

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.     

Protein crosslinking by transglutaminase controls cuticle morphogenesis in Drosophila

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins--two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin-as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.     

Mutations targeting intermodular interfaces or calcium binding destabilize the thrombospondin-2 signature domain

Thrombospondins (THBSs) are a family of secreted calcium-binding glycoproteins with roles in angiogenesis, cell motility, apoptosis, cytoskeletal organization, and extracellular matrix organization. The THBS-2 signature domain (three epidermal growth factor (EGF)-like modules, a wire module with 13 calcium-binding repeats, and a lectin-like module) binds 30 calcium ions and forms extensive interactions among its parts. We explored the significance of these structural elements by examining the impact of 10 different mutations known to result in pseudoachondrodysplasia or multiple epiphyseal dysplasia when found in the homologous wire and lectin-like modules of thrombospondin-5 (THBS-5). A variety of observations indicate that the mutations result in unstable THBS-5 proteins that aggregate in the endoplasmic reticulum. We introduced the mutations into homologous sites of a THBS-2 construct, for which the crystal structure is known, and determined the effects of the mutations on structure as assayed by differential scanning calorimetry and expression of the epitope for the 4B6.13 conformation-sensitive antibody. Abnormalities were found in one or more of several readouts: stability of interactions between the wire and lectin-like modules, stabilities of the EGF-like and wire modules, expression of the 4B6.13 epitope in soluble protein, and expression of the 4B6.13 epitope in substrate-adsorbed protein at different calcium concentrations. The patterns of abnormalities support the idea that the EGF-like, wire, and lectin-like modules constitute a dynamic and interactive calcium-sensitive structure in which a distortion at one site is transmitted to distal sites, leading to global changes in the protein.     

Boca-dependent maturation of beta-propeller/EGF modules in low-density lipoprotein receptor proteins

The extracellular portions of cell surface receptor proteins are often comprised of independently folding protein domains. As they are translated into the endoplasmic reticulum (ER), some of these domains require protein chaperones to assist in their folding. Members of the low-density lipoprotein receptor (LDLR) family require the chaperone called Boca in Drosophila or its ortholog, Mesoderm development, in the mouse. All LDLRs have at least one six-bladed beta-propeller domain, which is immediately followed by an epidermal growth factor (EGF) repeat. We show here that Boca is specifically required for the maturation of these beta-propeller/EGF modules through the secretory pathway, but is not required for other LDLR domains. Protein interaction data suggest that as LDLRs are translated into the ER, Boca binds to the beta-propeller. Subsequently, once the EGF repeat is translated, the beta-propeller/EGF module achieves a more mature state that has lower affinity for Boca. We also show that Boca-dependent beta-propeller/EGF modules are found not only throughout the LDLR family but also in the precursor to the mammalian EGF ligand.     

Binding of calcium to anticoagulant protein S: role of the fourth EGF module

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.     

EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X.

Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca(2+)-binding consensus sequence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca(2+)-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca(2+) affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca(2+) affinity, we expressed the Ca(2+)-binding EGF pair 3-4 from human protein S. Correct folding was shown by (1)H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by (1)H-(15)N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca(2+) binding were 1.0x10(-6) M and 4. 8x10(-3) M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca(2+) affinity of EGF 4, in the pair 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.     

Genetic Studies of Unusual Loci That Affect Body Shape of the Nematode CAENORHABDITIS ELEGANS and May Code for Cuticle Structural Proteins

In Caenorhabditis elegans, four loci (sqt-1, sqt-2, sqt-3 and rol-8) in which mutations affect body shape and cuticle morphology have unusual genetic properties. Mutant alleles of sqt-1 can interact to produce animals with a variety of mutant phenotypes: left roller, right roller, dumpy and long. At least three mutant phenotypes are specified by mutations in the sqt-3 locus. Most alleles at these loci are either dominant or cryptic dominant (i.e., are dominant only in certain genetic backgrounds). Most alleles of these loci exhibit codominance. Two putative null alleles of the sqt-1 locus produce a wild-type phenotype. Many alleles of these genes demonstrate unusual intergenic interactions that are not the result of simple epistasis: animals doubly heterozygous for mutations at two loci often display unexpected and unpredictable phenotypes. We suggest that these genetic properties might be expected of genes, such as the collagen genes, the products of which interact to form the animal's cuticle, and which are member genes of a gene family.     

The interaction of recombinant subdomains of the procollagen C-proteinase with procollagen I provides a quantitative explanation for functional differences between the two splice variants, mammalian tolloid and bone morphogenetic protein 1

The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease-CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphogenetic protein 1 (BMP-1, i.e., PCP-1) ends after the CUB3 domain. Two related genes encode proteases similar to mTld in humans and have been termed mammalian tolloid like-1 and -2 (mTll-1 and mTll-2, respectively). For mTll-1, it has been shown that it has C-proteinase activity. We demonstrate that recombinant EGF1-CUB3, CUB3, CUB3-EGF2, EGF2-CUB4, and CUB4-CUB5 modules of the procollagen C-proteinase can be expressed in bacteria and adopt a functional antiparallel beta-sheet conformation. As shown by surface plasmon resonance analysis, the modules bind to procollagen I in a 1:1 stoichiometry with dissociation constants (K(D)) ranging from 622.0 to 1.0 nM. Their binding to mature collagen I is weaker by at least 1 order of magnitude. Constructs containing EGF domains bind more strongly than those consisting of CUB domains only. This suggests that a combination of CUB and EGF domains serves as the minimal functional unit. The binding affinities of the EGF-containing modules for procollagen increase in the order EGF1-CUB3 < CUB3-EGF2 < EGF2-CUB4. In the context of the full length PCP, this implies that a given module has an affinity that continues to increase the more C-terminally the module is located within the PCP. The tightest binding module, EGF2-CUB4 (K(D) = 1.0 nM), is only present in mTld, which might provide a quantitative explanation for the different efficiencies of BMP-1 and mTld in procollagen C-proteinase activity.     

Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules

Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (>40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process.     

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides

Vitamin K-dependent protein S, which is a cofactor for activated protein C and thus important for down-regulation of the coagulation cascade, contains several Ca(2+)-binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (EGF) modules of protein S is the smallest fragment that retains high-affinity Ca(2+) binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the (1)H, 15N and (13)C resonances of the module pair with one Ca(2+) bound in EGF 4. In addition, nearly complete assignments of the (1)H resonances of the isolated Ca(2+)-free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis-trans isomerisation of the peptide bond preceding Pro-168, and dimerisation. Analysis of weighted average secondary chemical shifts, (3)J(HNHalpha) coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti-parallel beta-sheet.     

Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.     

Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions.

The Ca(2+)-binding epidermal growth factor (cbEGF)-like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch-1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca(2+)-binding. We report the Ca(2+)-binding properties of EGF11-12 and EGF10-13 from human Notch-1 (hNEGF11-12 and hNEGF10-13), modules previously shown to support Ca(2+)-dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5''-Br2BAPTA and 5-NBAPTA, gave two binding constants for hNEGF11-12, Kd1 = 3.4 x 10(-5) M and Kd2 > 2.5 x 10(-4) M. The high-affinity site was found to be localized to hNEGF12. Titration of hNEGF10-13 gave three binding constants, Kd1 = 3.1 x 10(-6) M, Kd2 = 1.6 x 10(-4) M, and Kd3 > 2.5 x 10(-4) M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11-12 and hNEGF10-13 had 10 to 100-fold higher affinity than reported for EGF32-33 and EGF25-31, respectively, from fibrillin-1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11-12 based on fibrillin-1 EGF32-33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca(2+)-binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.     

The epidermal growth factor precursor. A calcium-binding, beta-hydroxyasparagine containing modular protein present on the surface of platelets.

Various human body fluids and secretions contain a soluble form of the epidermal growth factor (EGF) precursor. The EGF precursor molecule contains eight EGF modules in addition to EGF itself. Using monoclonal antibodies specific for the EGF modules 7 and 8, we have purified the soluble form of the EGF precursor from human urine to homogeneity. The protein was shown to have a molecular mass of about 160 kDa and the N-terminal sequence SAPNHWSXPE. EGF modules 2, 7 and 8 of the precursor have the consensus sequence for post-translational beta-hydroxylation of Asp/Asn residues. We identified the presence of erythro-beta-hydroxy-aspartic acid (Hya) in acid hydrolysates of the EGF precursor (2.4 M.M protein-1). As the DNA sequence encodes Asn in the corresponding position, the Hya represents erythro-beta-hydroxyasparagine (Hyn). The Hyn-containing modules have a consensus calcium-binding motif immediately N-terminal of the first Cys residue. The synthetic EGF module 2 (residues 356-395) of the EGF precursor was found to bind calcium with low affinity, Kd approximately 3.5 mM, i.e. similar to the affinity of other isolated calcium-binding EGF modules. EGF module 7, when part of the intact protein, was found to bind Ca2+ with a Kd approximately 0.2 microM, i.e. approximately 10(4)-fold higher than that of isolated EGF modules presumably due to the influence of neighboring modules. We have detected EGF precursor in platelet-rich plasma and demonstrated it to be associated to platelets. The platelets were found to have 30-160 EGF molecules each.     

MUP-4 is a novel transmembrane protein with functions in epithelial cell adhesion in Caenorhabditis elegans

Tissue functions and mechanical coupling of cells must be integrated throughout development. A striking example of this coupling is the interactions of body wall muscle and hypodermal cells in Caenorhabditis elegans. These tissues are intimately associated in development and their interactions generate structures that provide a continuous mechanical link to transmit muscle forces across the hypodermis to the cuticle. Previously, we established that mup-4 is essential in embryonic epithelial (hypodermal) morphogenesis and maintenance of muscle position. Here, we report that mup-4 encodes a novel transmembrane protein that is required for attachments between the apical epithelial surface and the cuticular matrix. Its extracellular domain includes epidermal growth factor-like repeats, a von Willebrand factor A domain, and two sea urchin enterokinase modules. Its intracellular domain is homologous to filaggrin, an intermediate filament (IF)-associated protein that regulates IF compaction and that has not previously been reported as part of a junctional complex. MUP-4 colocalizes with epithelial hemidesmosomes overlying body wall muscles, beginning at the time of embryonic cuticle maturation, as well as with other sites of mechanical coupling. These findings support that MUP-4 is a junctional protein that functions in IF tethering, cell-matrix adherence, and mechanical coupling of tissues.     

A common cross-species function for the double epidermal growth factor-like modules of the highly divergent plasmodium surface proteins MSP-1 and MSP-8

An understanding of structural and functional constraints on the C-terminal double epidermal growth factor (EGF)-like modules of merozoite surface protein (MSP)-1 and related proteins is of importance to the development of these molecules as malaria vaccines and drug targets. Using allelic replacement, we show that Plasmodium falciparum parasites can invade erythrocytes and grow efficiently in the absence of an MSP-1 protein with authentic MSP-1 EGF domains. In this mutant parasite line, the MSP-1 EGFs were replaced by the corresponding double EGF module from P. berghei MSP-8, the sequence of which shares only low identity with its MSP-1 counterpart. Hence, the C-terminal EGF domains of at least some Plasmodium surface proteins appear to perform the same function in asexual blood-stage development. Mapping the surface location of the few residues that are common to these functionally complementary EGF modules revealed the presence of a highly conserved pocket of potential functional significance. In contrast to MSP-8, an even more divergent double EGF module, that from the sexual stage protein PbS25, was not capable of complementing MSP-1 EGF function. More surprisingly, two chimeric double EGF modules comprising hybrids of the EGF domains from P. falciparum and P. chabaudi MSP-1 were also not capable of replacing the P. falciparum MSP-1 EGF module. Together, these data suggest that although the MSP-1 EGFs can accommodate extensive sequence diversity, there appear to be constraints that may restrict the simple accumulation of point mutations in the face of immune pressure in the field.