Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen.

INTRODUCTION: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera. METHODS: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. RESULTS: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too. CONCLUSION: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.

A novel calcium-binding peptide from Antarctic krill protein hydrolysates and identification of binding sites of calcium-peptide complex.

Trypsin was used for preparing peptides with high calcium-binding capacity from Antarctic krill. Hydroxyapatite chromatography (HAC), size-exclusion chromatography (SEC), and reversed phase high performance liquid chromatography (RP-HPLC) were used to capture and purify calcium-binding peptides. The peptide sequence was determined to be VLGYIQIR (N- to C-terminal, MW = 960.58 Da), using LTQ Orbitrap XL. According to the results of FTIR and mass spectrometry, chelating site of calcium ions may possibly involve the carbonal or amino groups of Gln, Ile and Arg residues. Molecular dynamic simulation showed the conformation of peptide was markedly varied, and the distance between calcium ion and Gln and Ile residues was changing all the time. However, the distance between calcium ion and carboxyl oxygen of arginine residues was not changed significantly from 2 ns to 100 ns. Identified peptide can be used as a novel calcium supplement.

A novel antilithiatic protein from Tribulus terrestris having cytoprotective potency.

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.

Beneficial effect of teleost fish bone peptide as calcium supplements for bone mineralization.

A most common and trusted source of Ca is milk or other dairy products. However, some oriental people do not drink milk due to lactose indigestion and intolerance, which make them allergic to milk. There have been many studies on alternative calcium-rich diet or Ca supplements. Among them, teleost fish like anchovy and mola, which are commonly consumed in Asian countries, could be an important Ca dietary supplement, especially in population groups with low intakes of milk and dairy products. In this chapter, we summarize beneficial effects of teleost fish bone peptide (FBP) for Ca bioavailability and bone mineralization, based on our researches.

Heightened stability of polcalcin Phl p 7 is correlated with strategic placement of apolar residues.

Phl p 7 exhibits atypical conformational stability and a diminutive denaturational heat capacity increment, ΔC(p). Because exposure of apolar surface largely dictates the magnitude of ΔC(p), a depressed value could signify an unusually compact unfolded state. The volume of the denatured state ensemble (DSE) is evidently inversely correlated with mean hydrophobicity [Pace et al., Protein Sci. 19 (2010) 929-943]. Interestingly, apolar residues replace more polar ones at four positions in Phl p 7. We herein examine the consequences of replacing those residues with the corresponding ones from Bra n 1, a related isoform. All four mutations - M4H, L21A, I60T, and C63A - destabilize Phl p 7. Our analysis suggests that the DSE of Phl p 7 is indeed highly compact and that the substitutions act by increasing its volume and solvent-accessibility. All four mutations increase the urea m value; L21A, I60T, and C63A also yield a perceptible increase in ΔC(p).

Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen.

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

BACKGROUND: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. METHODS: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. RESULTS: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. CONCLUSIONS: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss.

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of mu-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the approximately 26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout mu-calpain and calpastatin were only partly purified; identity of trout mu-calpain was confirmed by labeling with antibodies to bovine skeletal muscle mu-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle mu- and m-calpain. Trout mu-calpain requires 4.4+/-2.8 microM and trout m-calpain requires 585+/-51 microM Ca(2+) for half-maximal activity, similar to the Ca(2+) requirements of mu- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.

An analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to its homologs in the phylum Platyhelminthes.

A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.

Essential role of Ca2+-binding protein 4, a Cav1.4 channel regulator, in photoreceptor synaptic function.

CaBP1-8 are neuronal Ca(2+)-binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4(-/-) mice than in control mice. Cabp4(-/-) retinas also had ectopic synapses originating from rod bipolar and horizontal cells tha HJt extended into the outer nuclear layer. Responses of Cabp4(-/-) rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4(-/-) mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Ca(v)1.4 alpha(1)-subunit and shifted the activation of Ca(v)1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca(2+) influx and neurotransmitter release in photoreceptor synaptic terminals.

Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata).

The mollusc shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are believed to play important roles in shell formation. In the present study, we extracted and purified a novel matrix protein, named Prismalin-14, from the acid-insoluble fraction of the prismatic layer of the shell of the Japanese pearl oyster (Pinctada fucata), and determined its whole amino acid sequence by a combination of amino acid sequence analysis and MS analysis of the intact protein and its enzymic digests. Prismalin-14 consisted of 105 amino acid residues, including PIYR repeats, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed inhibitory activity on calcium carbonate precipitation and calcium-binding activity in vitro. The scanning electron microscopy images revealed that Prismalin-14 affected the crystallization of calcium carbonate in vitro. A cDNA encoding Prismalin-14 was cloned and its expression was analysed. The amino acid sequence deduced from the nucleotide sequence of Prismalin-14 cDNA was identical with that determined by peptide sequencing. Northern-blot analysis showed that a Prismalin-14 mRNA was expressed only at the mantle edge. In situ hybridization demonstrated that a Prismalin-14 mRNA was expressed strongly in the inner side of the outer fold of the mantle. These results suggest that Prismalin-14 is a framework protein that plays an important role in the regulation of calcification of the prismatic layer of the shell.

Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves.

A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed.

Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution.

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.

Purification of matrix Gla protein from a marine teleost fish, Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5'rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted to regions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation.

Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor.

Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. We show that Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. We further show that the ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. Our results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels.

Olive pollen allergen Ole e 8: identification in mature pollen and presence of Ole e 8-like proteins in different pollens.

In a first approach, Ole e 8, a novel Ca2+-binding protein from olive pollen, was cloned and produced in Escherichia coli. We have obtained the natural form of Ole e 8 (nOle e 8) from the pollen and examined its immunologic equivalence with its recombinant form (rOle e 8). Size exclusion chromatography and a phenyl-Sepharose CL-4B affinity column were used to obtain nOle e 8 from the olive pollen. Inhibition assays by immunoblotting, using rOle e 8-specific rabbit antiserum, were performed to analyze the immunologic equivalence between the natural and the recombinant allergen, as well as to detect its presence in other pollens. Recombinant and natural Ole e 8 resulted immunologically equivalents, since they completely inhibited the IgG binding of the polyclonal antiserum to each other. Ole e 8-like proteins were detected in Oleaceae and Juniperus communis pollen, and might contribute to cross-reactivity processes between taxonomically related pollens.

Calreticulin enriched as an early-stage encapsulation protein in wax moth Galleria mellonella larvae.

To investigate the molecular mechanism of the early-stage encapsulation reaction in insects, we purified a 47kDa protein from injected beads into Galleria mellonella larvae. When a cDNA clone was isolated, the 47kDa protein showed high homology with Drosophila and human calreticulin. Western blotting analysis showed that the 47kDa protein was present in the hemocytes, but not in the plasma. When the early-stage encapsulated beads were coated with 47kDa protein antibody and reinjected into G. mellonella larvae, any further encapsulation reaction was inhibited. These results suggest that calreticulin is involved in non-self recognition in invertebrate cellular defense reactions.

Matrix Gla protein in Xenopus laevis: molecular cloning, tissue distribution, and evolutionary considerations.

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins and in higher vertebrates, is found in the extracellular matrix of mineralized tissues and soft tissues. MGP synthesis is highly regulated at the transcription and posttranscription levels and is now known to be involved in the regulation of extracellular matrix calcification and maintenance of cartilage and soft tissue integrity during growth and development. However, its mode of action at the molecular level remains unknown. Because there is a large degree of conservation between amino acid sequences of shark and human MGP, the function of MGP probably has been conserved throughout evolution. Given the complexity of the mammalian system, the study of MGP in a lower vertebrate might be advantageous to relate the onset of MGP expression with specific events during development. Toward this goal, MGP was purified from Xenopus long bones and its N-terminal amino acid sequence was determined and used to clone the Xenopus MGP complementary DNA (cDNA) by a mixture of reverse-transcription (RT)- and 5'- rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR). MGP messenger RNA (mRNA) was present in all tissues analyzed although predominantly expressed in Xenopus bone and heart and its presence was detected early in development at the onset of chondrocranium development and long before the appearance of the first calcified structures and metamorphosis. These results show that in this system, as in mammals, MGP may be required to delay or prevent mineralization of cartilage and soft tissues during the early stages of development and indicate that Xenopus is an adequate model organism to further study MGP function during growth and development.

Modulation of A-type potassium channels by a family of calcium sensors.

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor.

We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.