Low-density Lipoprotein Receptor Class Research Articles

Molecular cloning and expression of chitin deacetylase 1 gene from the gills of Penaeus monodon (black tiger shrimp).

Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P.monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P.monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp.

Identification and expression profiles of chitin deacetylase genes in the rice leaf folder, Cnaphalocrocis medinalis

Chitin deacetylase (CDA) is an insect chitin degradation enzyme that catalyzes the deacetylation of chitin to form chitosan. In this study, combination of rapid-amplification of cDNA ends (RACE) technology with Cnaphalocrocis medinalis transcriptome database analysis revealed the presence of at least five C. medinalis CDAs (CmCDAs), which were CmCDA1, CmCDA2, CmCDA4, CmCDA5, and CmCDA6. The cDNA sequences of CmCDA1, CmCDA2, and CmCDA4 had whole open reading frame (ORF) for further analysis. Phylogenetic analysis indicated that insect CDAs could be categorized into five groups. CmCDAs' structural domain analysis revealed that all three CDAs contained the chitin deacetylase-like catalytic domain. CmCDA1 and CmCDA2 belong to Group I because they both contain the chitin-binding peritrophin-A domain (ChBD), low-density lipoprotein receptor class A domain (LDLa), and chitin deacetylase-like catalytic domain. CmCDA4 only contains ChBD and chitin deacetylase-like catalytic domain thus belongs to Group III. Tissue and developmental stage expression analysis showed that the expression levels of CmCDA1, CmCDA2, and CmCDA4 are significantly higher in the head than other tissues and also significantly higher in adults than in larvae. CmCDA5 had significantly higher expression in the integument than other tissues, suggesting potential roles in the process of degradation of chitin. In contrast, CmCDA5 showed relatively high expression in larvae. In conclusion, this study analyzed the cDNA sequences of three CDA genes and determined their expression and molecular characteristics, which provided a new sequence resource and improved the development of bio-pesticides and the biological pest control and contributed to management of this important agricultural pest.

Identification of a homozygous missense mutation in LRP2 and a hemizygous missense mutation in TSPYL2 in a family with mild intellectual disability.

Non-syndromic autosomal recessive intellectual disability (ID) is a genetically heterogeneous disorder with more than 50 mutated genes to date. ID is characterized by deficits in memory skills and language development with difficulty in learning, problem solving, and adaptive behaviors, and affects ∼ 1% of the population. For detection of disease-causing mutations in such a heterogeneous disorder, homozygosity mapping together with exome sequencing is a powerful approach, as almost all known genes can be assessed simultaneously in a high-throughput manner. In this study, a hemizygous c.786C>G:p.Ile262Met in the testis specific protein Y-encoded-like 2 (TSPYL2) gene and a homozygous c.11335G>A:p.Asp3779Asn in the low-density lipoprotein receptor-related protein 2 (LRP2) gene were detected after genome-wide genotyping and exome sequencing in a consanguineous Pakistani family with two boys with mild ID. Mutations in the LRP2 gene have previously been reported in patients with Donnai-Barrow and Stickler syndromes. LRP2 has also been associated with a 2q locus for autism (AUTS5). The TSPYL2 variant is not listed in any single-nucleotide polymorphism databases, and the LRP2 variant was absent in 400 ethnically matched healthy control chromosomes, and is not listed in single-nucleotide polymorphism databases as a common polymorphism. The LRP2 mutation identified here is located in one of the low-density lipoprotein-receptor class A domains, which is a cysteine-rich repeat that plays a central role in mammalian cholesterol metabolism, suggesting that alteration of cholesterol processing pathway can contribute to ID.

A novel C-type lectin is involved in the innate immunity of Macrobrachium nipponense.

C-type lectins (CTLs) play important roles in invertebrate innate immunity by recognizing and eliminating pathogens. In the present study, a low-density lipoprotein receptor class A (LDLa) domain-containing CTL was identified from the oriental river prawn Macrobrachium nipponense, designated as MnCTLDcp1. The full-length cDNA of MnCTLDcp1 was composed of 1462 bp, with a 999-bp ORF encoding a 332-aa protein. An LDLa and a single C-type lectin-like domain (CTLD) were found. The mRNA transcripts of MnCTLDcp1 was expressed the highest in heart. After the prawns were challenged by Aeromonas hydrophila and Staphylococcus aureus, the expression level of MnCTLDcp1 in heart and hemocytes were all significantly up-regulated. Sugar binding assay revealed that the MnCTLDcp1 could bind to the glycoconjugates of bacteria surface, such as LPS, PGN and they can compete with bacterial as competitors. The recombinant MnCTLDcp1 agglutinates Gram-positive (S. aureus and Bacillus subtilis) and Gram-negative bacteria (A. hydrophila, Vibrio parahaemolyticus, Escherichia coli and Pseudomonas aeruginosa) in the presence of calcium and also could bind to these bacteria. These results clearly suggested that MnCTLDcp1 functions as a pattern-recognition receptor involved in the innate immunity of M. nipponense.

Isolation and characterization of two novel C-type lectins from the oriental river prawn, Macrobrachium nipponense.

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins which are believed to play important roles in the innate immunity of invertebrates. This study identified two novel C-type lectins, designated as MnCTLDcp2 and MnCTLDcp3, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnCTLDcp2 was of 1582 bp with an open reading frame (ORF) of 972 bp encoding a polypeptide of 323 amino acids. The complete nucleotide sequence of MnCTLDcp3 cDNA was 583 bp, containing a 555 bp ORF encoding a putative protein of 184 deduced amino acids. The deduced MnCTLDcp2 and MnCTLDcp3 proteins both contained a single C-type lectin-like domain (CTLD). Besides, MnCTLDcp2 contains a signal peptide and an low-density lipoprotein receptor class A (LDLa) domain. Reverse transcription PCR showed that MnCTLDcp2 was expressed in the heart, gill, nerve hepatopancreas and intestine; MnCTLDcp3 was expressed in the hepatopancreas, heart, nerve, gill and muscle. Their expression in the heart tissue was regulated following challenge with bacteria. The microbial agglutination assay showed that both MnCTLDcp2 and MnCTLDcp3 could agglutinated bacteria in the presence of calcium. All these results suggested that MnCTLDcp2 and MnCTLDcp3 functioned as pattern recognition receptors in the immune system of M. nipponense.

Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study.

BackgroundThe metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas.MethodsWe classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis.ResultsGroup-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported.ConclusionsIntratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1469-5) contains supplementary material, which is available to authorized users.

Molecular characterization and RNA interference analysis of vitellogenin receptor from Nilaparvata lugens (Stål)

Vitellogenin receptors (VgRs), members of the low-density lipoprotein receptor (LDLR) superfamily, are responsible for taking vitellogenin (Vg) into developing oocytes. Here the first full-length VgR cDNA from a hemipteran insect, the brown planthopper (Nilaparvata lugens), was cloned and sequenced. The complete mRNA sequence was 6174bp in length with an open reading frame (ORF) of 5796bp encoding 1931 amino acid residues. N. lugens VgR (NlVgR) contained two ligand-binding domains with five LDLR Class A cysteine-rich repeats in the first domain and eight in the second domain, which was similar to other insect VgRs. NlVgR was specifically expressed in the ovary, and the mRNA level started to increase after adult female emergence, with a peak on day 7 in the adult stage, and then declined. Western blot analysis of NlVgR protein revealed an ovary-specific expression pattern, which was consistent with NlVgR transcript detection. Injection with NlVgR double-stranded RNA (dsRNA) significantly disturbed NlVgR, which led to a decrease in NlVg protein content in the ovaries, an accumulation of NlVg protein in the hemolymph, the arrested development of ovaries, and the failure of insects to reproduce. Besides, NlVgR expression was significantly upregulated after the topical application of juvenile hormone (JH) III. These results suggest that VgR is critical for Vg uptaking of oocytes and it plays an important role in insect fecundity.

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia.

Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.

Cloning and Tissue-Specific Expression of a Chitin Deacetylase Gene from Helicoverpa armigera (Lepidoptera: Noctuidae) and Its Response to Bacillus thuringiensis.

Chitin deacetylases (CDAs) convert chitin into chitosan, the N-deacetylated form of chitin, which influences the mechanical and permeability properties of structures such as the cuticle and peritrophic matrices. In this article, a new CDA encoding gene, Hacda2, was cloned by reverse transcription-polymerase chain reaction method in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), with an open reading frame of 1,611 bp. The deduced protein composed of 536 amino acid residues with a signal peptide, a chitin-binding domain, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase-like catalytic domain. The highest expression level of Hacda2 was detected in fat body among tissues tested in the fifth-instar larvae using real-time quantitative polymerase chain reaction method. Feeding of Bacillus thuringiensis (Bt) (Bacillales: Bacillaceae) diet changed the expression level of Hacda1, Hacda2, Hacda5a, and Hacda5b significantly and differentially in the third-instar larvae. Hacda5a and Hacda5b expression were initially down-regulated and then up-regulated, whereas, the expression level of Hacda1 and Hacda2 was suppressed constantly postfeeding on Bt diet. These results suggested that HaCDAs may be involved in the response of H. armigera larvae to Bt and may be helpful to elucidate the roles of HaCDAs in the action of Bt cry toxin. The potential of HaCDAs to be used as synergists of Bt insecticidal protein needs to be further tested.

Functional characterization and classification of frequent low-density lipoprotein receptor variants.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder mostly caused by mutations in the low-density lipoprotein receptor (LDLR) gene leading to increased risk for premature cardiovascular diseases. According to functional studies, LDLR mutations may be classified into five classes. The main objective of this study was to characterize seven LDLR variants previously detected in FH patients. Analysis by flow cytometry and confocal microscopy of LDLR activity demonstrate that all the studied variants are pathogenic. Among the mutations located in β-propeller, p.Trp577Gly and p.Ile624del were classified as class 2, whereas p.Arg416Trp and p.Thr454Asn as class 5. p.Phe800Glyfs*129 (located in the cytoplasmic domain), p.Cys155Tyr (located in the binding domain), and p.Asn825Lys (inside FxNPxY motif) were classified as class 2, 3, and 4, respectively. The results also show that LDLR activity of these class 4 and 5 variants is not completely abolished, showing a milder phenotype. We have also determined that statin response is more efficient lowering total cholesterol in heterozygous patients carrying p.Ile624del (class 2) compared with p.Arg416Trp and p.Thr454Asn (class 5) variants. In conclusion, these findings emphasize the importance of characterizing LDLR pathogenic variants to provide an indisputable FH diagnosis and to gain insight into the statin response depending on the LDLR class mutation.

Learning from biology: synthetic lipoproteins for drug delivery.

Synthetic lipoproteins represent a relevant tool for targeted delivery of biological/chemical agents (chemotherapeutics, siRNAs, photosensitizers, and imaging contrast agents) into various cell types. These nanoparticles offer a number of advantages for drugs delivery over their native counterparts while retaining their natural characteristics and biological functions. Their ultra-small size (<30 nm), high biocompatibility, favorable circulation half-life, and natural ability to bind specific lipoprotein receptors, i.e., low-density lipoprotein receptor (LDLR) and Scavenger receptor class B member 1 (SRB1) that are found in a number of pathological conditions (e.g., cancer, atherosclerosis), make them superior delivery strategies when compared with other nanoparticle systems. We review the various approaches that have been developed for the generation of synthetic lipoproteins and their respective applications in vitro and in vivo. More specifically, we summarize the approaches employed to address the limitation on use of reconstituted lipoproteins by means of natural or recombinant apolipoproteins, as well as apolipoprotein mimetic molecules. Finally, we provide an overview of the advantages and disadvantages of these approaches and discuss future perspectives for clinical translation of these nanoparticles.

Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

The low density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the "SimpleCell" O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position -1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.

Functional Characterization of Two Low-Density Lipoprotein Receptor Gene Mutations in Two Chinese Patients with Familial Hypercholesterolemia

BackgroundFamilial hypercholesterolemia (FH) is an autosomal dominant disease that primarily results from mutations in the low-density lipoprotein receptor (LDLR) gene. We investigated two unrelated Chinese FH patients using gene screening and functional analysis to reveal the pathogenicity and the mechanism by which these mutations cause FH.MethodsFirst, the LDLR gene was sequenced in these patients. Then, mutant receptors were transfected into human embryo kidney 293(HEK-293) cells, and a confocal laser-scanning microscope was used to observe the localization of mutant proteins. Further, the expression and the internalization activity were analyzed by flow cytometry. Finally, LDLR protein expression and stability was detected by western blot.ResultsTwo different LDLR class 2B mutations were detected in two patients. The C201F mutation is a known mutation. However, the G615V mutation is novel. Flow cytometry showed that the expression and internalization activity of the mutant LDLRs were reduced to 73.6% and 82.6% for G615V and 33.2% and 33.5% for C201F, respectively.ConclusionsThis study identified two LDLR mutations in Chinese patients with FH and analyzed the relationship between the genotype and phenotype of these patients. We found that these mutant LDLRs were defective in transport, which led to a reduction in cholesterol clearance. These results increase our understanding of the mutational spectrum of FH in the Chinese population.

An LDLa domain-containing C-type lectin is involved in the innate immunity of Eriocheir sinensis

C-type lectins (CTLs) have crucial functions in recognizing and eliminating pathogens in innate immunity. This study identified a novel low-density lipoprotein receptor class A (LDLa) domain-containing CTL, designated as EsCTLDcp, from the Chinese mitten crab Eriocheir sinensis. The EsCTLDcp cDNA is 1258bp long, with a 975bp open reading frame that encodes a 324-amino acid protein. EsCTLDcp contains a signal peptide, an LDLa, and a single C-type lectin-like domain. EsCTLDcp was only expressed in the hepatopancreas of normal crabs, and its expression was regulated following crab challenge with pathogen-associated molecular patterns and with bacteria. The recombinant EsCTLDcp agglutinates Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. rEsCTLDcp also binds to various bacteria including S. aureus, Bacillus thuringiensis, Bacillus subtilis, Escherichia coli, Vibrio natriegens, V. parahaemolyticus, and A. hydrophila. The rEsCTLDcp protein helped the crabs clear the virulent Gram-negative bacterium V. parahaemolyticus in vivo, as well as interacted with VP24, an envelope protein of white spot syndrome virus (WSSV). These data suggest that EsCTLDcp functions as a pattern-recognition receptor involved in the innate immunity of E. sinensis.

Two novel C-type lectins with a low-density lipoprotein receptor class A domain have antiviral function in the shrimp Marsupenaeus japonicus

C-type lectins (CTLs) are pattern-recognition receptors (PRRs) that play important roles in immune response. In this study, two new CTLs containing a low-density lipoprotein receptor class A domain (LDLR) and a carbohydrate recognition domain (CRD) were identified in Marsupenaeus japonicus and designated as LdlrLec1 and LdlrLec2. The two CTLs expressed in all the tested tissues of shrimp, however, LdlrLec1 was mainly expressed in hemocytes, heart, gill and intestines, whereas LdlrLec2 was expressed in hepatopancreas and heart. The expression patterns of both LdlrLec1 and LdlrLec2 mRNA were obviously upregulated upon white spot syndrome virus (WSSV) challenge. Injection of recombinant LdlrLec1 or LdlrLec2 into shrimp inhibited WSSV replication, whereas knocking down the expression of LdlrLec1 and LdlrLec2 by RNA interference increased WSSV replication in vivo. The infection rates of WSSV incubated with LdlrLecs were reduced significantly compared with the control group. The LdlrLec proteins could interact with VP28, a major envelope protein of WSSV, which is necessary for the attachment and penetration of WSSV into shrimp cells. These results indicate that LdlrLec1 and LdlrLec2 may function in antiviral response by binding to WSSV and inhibiting their pervasion and replication in shrimp.

Cloning and molecular characterization of complement component 1 inhibitor (C1INH) and complement component 8β (C8β) in Nile tilapia (Oreochromis niloticus)

Nile tilapia (Oreochromis niloticus), one of the most important groups of food fishes in the world, has frequently suffered from serious challenge from pathogens in recent years. Immune responses of Nile tilapia should be understood to protect the aquaculture industry of this fish. The complement system has an important function in recognizing bacteria, opsonizing these pathogens by phagocytes, or killing them by direct lysis. In this study, two Nile tilapia complement component genes, complement component 1 inhibitor (C1INH) and complement component 8β subunit (C8β), were cloned and their expression characteristics were analyzed. C1INH cDNA was found containing a 1791 bp open reading frame (ORF) encoding a putative protein with 597 amino acids, a 101 bp 5′-untranslated region (UTR) and a 236 bp 3′-UTR. The predicted protein structure for this gene consisted of two Ig-like domains and glycosyl hydrolase family-9 active site signature 2. The C8β cDNA consisted of a 1761 bp ORF encoding 587 amino acids, a 15 bp 5′-UTR and a 170 bp 3′-UTR. The predicted protein of C8β contained three motifs, thrombospondin type-1 repeat, membrane attack complex/perforin domain, and LDL-receptor class A. Expression analysis revealed that these two complement genes were highly expressed in the liver, however, were weakly expressed in the gill, heart, brain, kidney, intestine, spleen and dorsal muscle tissues. The present study provided insights into the complement system and immune functions of Nile tilapia.

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.

Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation

Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein domains (CUB repeat) and four low-density lipoprotein receptor class A domains (LDLRA repeat). The single-chain zymogen of matriptase has been found to exhibit substantial protease activity, possibly causing its own activation (i.e. conversion to a disulfide-linked two-chain fully active form), although the activation seems to be mediated predominantly by two-chain molecules. Our aim was to assess the roles of CUB and LDLRA repeats in zymogen activation. Transient expression studies of soluble truncated constructs of recombinant matriptase in COS-1 cells showed that the CUB repeat had an inhibitory effect on zymogen activation, possibly because it facilitated the interaction of two-chain molecules with a matriptase inhibitor, hepatocyte growth factor activator inhibitor type-1. By contrast, the LDLRA repeat had a promoting effect on zymogen activation. The effect of the LDLRA repeat seems to reflect its ability to increase zymogen activity. The proteolytic activities were higher in pseudozymogen forms of recombinant matriptase containing the LDLRA repeat than in a pseudozymogen without the repeat. Our findings provide new insights into the roles of these non-catalytic domains in the generation of active matriptase.

The SOL-2/Neto Auxiliary Protein Modulates the Function of AMPA-Subtype Ionotropic Glutamate Receptors

The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

Crystal Structure of C5b-6 Suggests Structural Basis for Priming Assembly of the Membrane Attack Complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12-18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.