The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family

Several cDNA clones with similarity to α-amylases have been characterized from a library made from adult female salivary gland RNA isolated from the vector mosquito, Aedes aegypti. The corresponding gene, designated Amylase I (Amy I), is expressed specifically in the proximal-lateral lobes of the adult female salivary gland, a pattern overlapping that of another gene, Mal I, involved in carbohydrate metabolism. The deduced amino acid sequence of Amy I indicates that this gene encodes a protein, approximate M_r= 81,500, that appears to be a novel member of the amylase gene family. The mosquito protein contains a putative signal peptide for secretion and several consensus sites for asparagine-linked glycosylation. The Amy I protein shows significant similarity to invertebrate and vertebrate amylases including the conservation of four reactive and substrate binding sites. However, the amino-terminal region of the Amy-I protein is unique to the mosquito. Similarity with the Drosophila melanogaster protein is evident only after the first 260 amino acids in the mosquito sequence. The identification of this gene and its expression pattern adds to the observed relationship between spatial-specific gene expression in the female salivary glands and the specific feeding mode of the adult mosquito.     

Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.     

MAEBL is essential for malarial sporozoite infection of the mosquito salivary gland

Malarial sporozoites mature in the oocysts formed in the mosquito midgut wall and then selectively invade the salivary glands, where they wait to be transmitted to the vertebrate host via mosquito bite. Invasion into the salivary gland has been thought to be mediated by specific ligand-receptor interactions, but the molecules involved in these interactions remain unknown. MAEBL is a single transmembrane-like protein that is structurally related to merozoite adhesive proteins. We found MAEBL of the rodent malaria parasite, Plasmodium berghei, to be specifically produced by the sporozoites in the oocyst and localized in their micronemes, which are secretory organelles involved in malarial parasite invasion into the host cell. A targeted disruption experiment of the P. berghei MAEBL gene revealed that it was essential for sporozoite infection of the salivary gland and was involved in the attachment to the salivary gland surface. In contrast, the disruption of the MAEBL gene did not affect sporozoite motility in vitro nor infectivity to the vertebrate host. These results suggest that P. berghei MAEBL is a sporozoite attachment protein that participates in specific binding to and infection of the mosquito salivary gland.     

Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy.

The causes of arterial calcification are beginning to be elucidated. Macrophages, mast cells, and smooth muscle cells are the primary cells implicated in this process. The roles of a variety of bone-related proteins including bone morphogenetic protein-2 (BMP-2), matrix Gla protein (MGP), osteoprotegerin (OPG), osteopontin, and osteonectin in regulating arterial calcification are reviewed. Animals lacking MGP, OPG, smad6, carbonic anhydrase isoenzyme II, fibrillin-1, and klotho gene product develop varying extents of arterial calcification. Hyperlipidemia, vitamin D, nicotine, and warfarin, alone or in various combinations, produce arterial calcification in animal models. MGP has recently been discovered to be an inhibitor of bone morphogenetic protein-2, the principal osteogenic growth factor. Many of the forces that induce arterial calcification may act by disrupting the essential post-translational modification of MGP, allowing BMP-2 to induce mineralization. MGP requires gamma-carboxylation before it is functional, and this process uses vitamin K as an essential cofactor. Vitamin K deficiency, drugs that act as vitamin K antagonists, and oxidant stress are forces that could prevent the formation of GLA residues on MGP. The potential role of arterial apoptosis in calcification is discussed. Potential therapeutic options to limit the rate of arterial calcification are summarized.     

Storage and secretion of the peritrophic matrix protein Ag-Aper1 and trypsin in the midgut of Anopheles gambiae

The gene Ag-Aper1 encodes a peritrophic matrix (PM) protein from the mosquito Anopheles gambiae. Ag-Aper1 gene expression and protein localization in the mosquito midgut were studied during the course of a blood meal. Ag-Aper1 mRNA abundance does not change appreciably during the course of blood ingestion and digestion. Prior to a blood meal, the protein is stored in secretory vesicles of midgut epithelial cells. Moreover, Ag-Aper1 colocalizes to the same secretory vesicles as trypsin, indicating that these proteins use a common secretory pathway. Blood feeding triggers the secretion of vesicle contents into the midgut lumen, after which Ag-Aper1 is incorporated into the PM. Newly synthesized Ag-Aper1 protein was again detected within the midgut epithelial cells at 60 h after blood ingestion.     

Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification

BACKGROUND: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. METHODS AND RESULTS: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. CONCLUSIONS: These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.     

Developmental variation in epidermal growth factor receptor size and localization in the malaria mosquito, Anopheles gambiae

The AGER gene encoding the epidermal growth factor receptor (EGFR) of the malaria mosquito Anopheles gambiae was cloned and sequenced. It represents a canonical member of this family of tyrosine kinase proteins exhibiting many similarities to orthologues from other species, both on the level of genomic organization and protein structure. The mRNA can be detected throughout development. Western analysis with an antibody raised against the extracellular domain of the mosquito protein suggests developmental variation in protein size and location that may be involved in the function of EGFR in the mosquito.     

The local calcification inhibitor matrix Gla protein in pseudoxanthoma elasticum

OBJECTIVES: Recent studies have revealed the involvement of calcification inhibitory proteins in the pathogenesis of pseudoxanthoma elasticum (PXE). DESIGN AND METHODS: We analyzed serum concentrations of the calcification inhibitor matrix Gla protein (MGP) in a large cohort of patients suffering from PXE (n=101), 34 first-degree relatives and 67 healthy controls. Moreover, we determined the distribution of the two MGP promoter polymorphisms c.-7G>A and c.-138T>C in the three cohorts. RESULTS: We found significantly lower total MGP concentrations in the sera of PXE patients compared to healthy controls (p=0.0002). Furthermore, higher serum MGP concentrations could be correlated with a later PXE onset. Analysis of MGP promoter polymorphism frequencies revealed one MGP haplotype to be a potential protective co-factor in PXE. CONCLUSIONS: Our findings point to a role of the local calcification inhibitor MGP in PXE manifestation.     

Circulating matrix γ‐carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome

Summary. Background and objectives: Matrix γ‐carboxyglutamate protein (MGP), a vitamin K‐dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an opportunity to investigate the functions of MGP. The purpose of this study was (i) to investigate the phenotype and the underlying MGP mutation of a newly identified KS patient, and (ii) to investigate MGP species and the effect of vitamin K supplements in KS patients. Methods: The phenotype of a newly identified KS patient was characterized with specific attention to signs of vascular calcification. Genetic analysis of the MGP gene was performed. Circulating MGP species were quantified and the effect of vitamin K supplements on MGP carboxylation was studied. Finally, we performed immunohistochemical staining of tissues of the first KS patient originally described focusing on MGP species. Results: We describe a novel homozygous MGP mutation (c.61+1G>A) in a newly identified KS patient. No signs of arterial calcification were found, in contrast to findings in MGP knockout mice. This patient is the first in whom circulating MGP species have been characterized, showing a high level of phosphorylated MGP and a low level of carboxylated MGP. Contrary to expectations, vitamin K supplements did not improve the circulating carboxylated MGP levels. Phosphorylated MGP was also found to be present in the first KS patient originally described. Conclusions: Investigation of the phenotype and MGP species in the circulation and tissues of KS patients contributes to our understanding of MGP functions and to further elucidation of the difference in arterial phenotype between MGP‐deficient mice and humans.     

Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats

Summary.  The vitamin K-dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein-2 (BMP-2). Here we present additional evidence that the Ca²⁺-induced conformer of the vitamin K-dependent Gla region in MGP is involved in BMP-2 binding. Recombinant BMP-2 binds to the Gla-containing region of MGP in the presence of Ca²⁺. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly γ-carboxylated and did not bind BMP-2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP-2/MGP complex exists in vivo , consistent with a role for MGP as a BMP-2 inhibitor. Age-related arterial calcification may be a consequence of under-γ-carboxylation of MGP, allowing unopposed BMP-2 activity.     

High expression of genes for calcification-regulating proteins in human atherosclerotic plaques.

Calcification is common in atheromatous plaques and may contribute to plaque rupture and subsequent thrombosis. However, little is known about the mechanisms which regulate the calcification process. Using in situ hybridization and immunohistochemistry we show that two bone-associated proteins, osteopontin (OP) and matrix Gla protein (MGP), are highly expressed in human atheromatous plaques. High levels of OP mRNA and protein were found in association with necrotic lipid cores and areas of calcification. The predominant cell type in these areas was the macrophage-derived foam cell, although some smooth muscle cells could also be identified. MGP was expressed uniformly by smooth muscle cells in the normal media and at high levels in parts of the atheromatous intima. Highest levels of this matrix-associated protein were found in lipid-rich areas of the plaque. The pattern of expression of these two genes contrasted markedly with that of calponin and SM22 alpha, genes expressed predominantly by differentiated smooth muscle cells and whose expression was generally confined to the media of the vessel. The postulated function of OP and MGP as regulators of calcification in bone and the high levels and colocalization of both in atheromatous plaques suggest they have an important role in plaque pathogenesis and stability.     

Targeted disruption of the plasmodium berghei CTRP gene reveals its essential role in malaria infection of the vector mosquito

CTRP (circumsporozoite protein and thrombospondin-related adhesive protein [TRAP]-related protein) of the rodent malaria parasite Plasmodium berghei (PbCTRP) makes up a protein family together with other apicomplexan proteins that are specifically expressed in the host-invasive stage 1. PbCTRP is produced in the mosquito-invasive, or ookinete, stage and is a protein candidate for a role in ookinete adhesion and invasion of the mosquito midgut epithelium. To demonstrate involvement of PbCTRP in the infection of the vector, we performed targeting disruption experiments with this gene. PbCTRP disruptants showed normal exflagellation rates and development into ookinetes. However, no oocyst formation was observed in the midgut after ingestion of these parasites, suggesting complete loss of their invasion ability. On the other hand, when ingested together with wild-type parasites, disruptants were able to infect mosquitoes, indicating that the PbCTRP gene of the wild-type parasite rescued infectivity of disruptants when they heterologously mated in the mosquito midgut lumen. Our results show that PbCTRP plays a crucial role in malaria infection of the mosquito midgut and suggest that similar molecular mechanisms are used by malaria parasites to invade cells in the insect vector and the mammalian host.     

Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host

Sporozoites are an invasive stage of the malaria parasite in both the mosquito vector and the vertebrate host. We developed an in vivo assay for mosquito salivary gland invasion by preparing Plasmodium gallinaceum sporozoites from infected Aedes aegypti mosquitoes under physiological conditions and inoculating them into uninfected female Ae. aegypti. Sporozoites from mature oocysts were isolated from mosquito abdomens 10 or 11 d after an infective blood meal. Salivary gland sporozoites were isolated 13 or 14 d after an infective blood meal. Purified oocyst sporozoites that were inoculated into uninfected female mosquitoes invaded their salivary glands. Using the same assay system, sporozoites derived from salivary glands did not reinvade the salivary glands after inoculation. Conversely, as few as 10 to 50 salivary gland sporozoites induced infection in chickens, while only 2 of 10 chickens inoculated with 5,000 oocyst sporozoites were infected. Both sporozoite populations were found to express a circumsporozoite protein on the sporozoite surface as determined by immunofluorescence assay and circumsporozoite precipitation test using a circumsporozoite protein-specific monoclonal antibody. We conclude that molecules other than this circumsporozoite protein may be responsible for the differential invasion of mosquito salivary glands or infection of the vertebrate host.     

Sequence of a mosquito ribonucleotide reductase cDNA and evidence for gene amplification in hydroxyurea-resistant cells

Ribonucleotide reductase is essential for production of the deoxyribonucleotide substrates required for DNA synthesis in all eukaryotic cells. The full-length cDNA encoding a mosquito ribonucleotide reductase R2 subunit was obtained from Aedes albopictus cells using a polymerase chain reaction (PCR)-based approach. The cDNA contained 1197 nucleotides, and encoded a 398 amino acid R2 protein. Overall, mosquito R2 shared approximately 70% amino acid sequence identity with R2 proteins from vertebrates, but conservation at the N-terminus of the protein was relatively low. The sequence of R2 cDNA was identical in hydroxyurea-resistant mosquito cells and in wild-type cells, while the R2 gene copy number was increased ten to twentyfold in resistant cells.