Expression in Escherichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein

Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein. In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA. In addition, we isolated and purified to homogeneity, native rat brain calbindin D28. The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28. It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation. The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays. We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium. To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions). These deletions resulted in smaller proteins that still bound calcium. The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.     

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma-chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross-hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H-chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H-chains, a mechanism which is not operative in the chicken L-chain locus. The most notable among the chicken Igs is the so-called 7S IgG because its H-chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H-chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8-10 H-chain genes.     

Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution.     

Tissue-specific regulation of the concentration of calbindin D28K mRNA in the developing chicken

A BamHI-HindIII restriction fragment containing the 5'-terminal portion of the gene encoding chicken Calbindin D28K was sequenced and used as a probe in Northern-blot hybridizations to RNA extracted from the brain and intestine of chickens at various stages of development. In both tissues Calbindin D28K mRNA consists of a family of three species, which differ by size. In the intestine Calbindin D28K mRNAs appear at hatching and reach a peak at day 7. In the brain the same RNA species are easily detected at least 7 days before hatching, show a moderate increase at hatching and remain essentially constant during the first 10 days of adult life. The concentration of Calbindin D28K mRNAs in the intestine is strictly dependent on Vitamin D, while it is not in the cerebellum.     

cDNA Cloning of the Human Homologues of the MouseKe4andKe6Genes at the Centromeric End of the Human MHC Region

cDNA clones corresponding to theHKE4andHKE6genes at the centromeric end of the HLA region on human chromosome 6p21.3 were isolated and characterized. The predicted amino acid sequences of HKE4 and HKE6 exhibited 81.5 and 85.6% identity to the mouse homologues, Ke4 and Ke6, respectively.HKE4may encode a membrane protein with histidine-rich charge clusters. HKE6 possesses remarkable amino acid sequence conservation with several bacterial proteins with oxidoreductase function and also shows significant homology with the two unique functional domains containing the nucleotide cofactor binding site and the consensus motif characteristic of the members of the superfamily of short-chain alcohol dehydrogenases such as human and rat steroid and prostaglandin dehydrogenases.     

Molecular cloning of a multifunctional chicken protein acting as the prolyl 4-hydroxylase beta-subunit, protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. Comparison of cDNA-deduced amino acid sequences with those in other species.

Several recent studies indicate that a single polypeptide may act as the beta-subunit of prolyl 4-hydroxylase, the enzyme protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. We report here the isolation and characterization of cDNA clones encoding this multifunctional protein in the chicken. All the coding sequences were determined on the basis of nucleotide sequencing of five cDNA clones and amino acid sequencing of the N-terminal end of the chicken beta-subunit. The processed polypeptide contains 493 amino acid residues, the size of the respective mRNA being about 2.7 kb. The chicken beta-subunit cDNA sequences were 78% homologous to the previously reported human beta-subunit cDNA sequences at the nucleotide level and 85% homologous at the amino acid level. The homology of the chicken beta-subunit sequences to those reported for bovine thyroid-hormone-binding protein and rat protein disulphide-isomerase was also 85% at the amino acid level. Primary-structure comparisons between the four species indicated that the two proposed active sites of protein disulphide-isomerase, the two Trp-Cys-Gly-His-Cys-Lys sequences, are located within highly conserved regions, which are also homologous to the active sites of a number of thioredoxins. The middle of the polypeptide has an additional conserved region 100 amino acid residues in length in which the degree of homology between the four species is 94% at the amino acid level. This long conserved region may also be important for some of the multiple functions of the protein. The four extreme C-terminal amino acids of the polypeptide in all four species are Lys-Asp-Glu-Leu, a sequence that has been suggested to function as a signal for the retention of a protein in the endoplasmic reticulum.     

A single receptor identical with that from intestine/T47D cells mediates the action of 1,25-dihydroxyvitamin D-3 in HL-60 cells.

Two anti-vitamin D receptor monoclonal antibodies binding to two different epitopes immunoprecipitate 100% of the HL-60 1,25-dihydroxyvitamin D-3 binding activity, while another monoclonal antibody specific for the porcine receptor precipitates none. Using a rat receptor cDNA probe, a single mRNA species of 4.6 kb was detected by Northern analysis of HL-60 mRNA. Using a cDNA probe from the cloned rat receptor, 10(7) recombinants from a lambda gt11 cDNA library constructed from mRNA isolated from HL-60 cells was screened yielding two positive clones. These clones had sequences identical with the known human receptor sequence from intestinal/T47D sources. Using PCR technology, the entire sequence of the HL-60 1,25-dihydroxyvitamin D-3 receptor was determined. This sequence was found identical with that reported for the human intestinal/T47D cDNA encoding the vitamin D receptor except for a single base. The substitution of this particular base does not alter the amino acid sequence however. Thus, the same receptor likely operates in differentiation and calcium transport functions.     

Monoclonal antibodies directed against the calcium binding protein Calbindin D-28k

We have produced 25 clones secreting antibodies directed against chicken Calbindin D-28k. Two of them, 300 and 318, recognize determinants conserved in fish, chicken, mouse, rat, rabbit, monkey and human Calbindin D-28k. We demonstrate their use in the immunohistochemical localization of Calbindin D-28k, and in the detection of Calbindin D-28k on immunoblots.     

Structure of the human brain calcium-binding protein calretinin and its expression in bacteria

Calbindin D28k and calretinin are two closely related intracellular calcium-binding proteins belonging to the troponin C superfamily. Calbindin is known to be involved in the vitamin-D-dependent calcium absorption through intestinal and renal epithelia, while the function of neuronal calbindin and calretinin is poorly understood. Using antibodies directed against chick intestinal calbindin D28k, human calretinin cDNA clones were isolated from brain cDNA libraries. The sequence of the calretinin cDNA revealed an open reading frame of 271 codons coding for a protein of 31,520 Da, and sharing 58% identical residues with human calbindin D28k. Calretinin contains five presumably active and one presumably inactive calcium-binding domains. Comparison with the partial sequences available for chick and guinea pig calretinins revealed that the protein is highly conserved in evolution (evolutionary rate: 0.27 x 10(-9) amino acid-1 year-1). The calretinin message was detected in the brain, while absent from heart muscle, kidney, liver, lung, spleen, stomach and thyroid gland. Recombinant calretinin was expressed in Escherichia coli, and the calcium-binding properties were confirmed on both the natural and the recombinant proteins. Part of the human gene coding for calretinin was isolated and the region corresponding to the promoter and the first exon was sequenced.     

Rat vitamin D binding protein. Determination of the full-length primary structure from cloned cDNA

Vitamin D binding protein (DBP) is an abundant serum glycoprotein secreted by the liver which transports vitamin D sterols, binds to actin, and is found on the surface of B-lymphocytes and subpopulations of T-lymphocytes. In the current study, a cDNA to rat DBP mRNA was cloned from a bacteriophage lambda gt 11 rat liver expression library. This DBP cDNA clone was identified by immunoblotting and its identity was confirmed by immunoprecipitation of a 54-kDa protein after hybrid-assisted translation. Northern analysis and primer extension mapping of rat liver mRNA indicated that the full-length DBP mRNA contains 1700 bases. By DNA sequence analysis this 1655-base pair clone contains a single open reading frame encoding the 476-amino acid containing full-length DBP and includes its 16-amino acid signal sequence. Analysis of the sequence reveals about 40% nucleotide and 23% amino acid homology to both albumin and alpha-fetoprotein. The encoded DBP contains a characteristic placement of cysteine residues, identical to that in albumin, suggesting a similar secondary folding structure. Albumin and alpha-fetoprotein are composed of three internally homologous domains. DBP mRNA terminates 122 amino acids before the larger albumin mRNA in the third internal domain, but retains the characteristic homology among the first two domains and the truncated portion of the third domain. These data support the conclusion that DBP is a member of a multigene family which includes albumin and alpha-fetoprotein.     

Molecular cloning of the rat stomach (H+ + K+)-ATPase

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.     

Expression and purification of human calbindin D28k

Calbindin D28k is a protein abundant in the mammalian central nervous system and in epithelial tissue involved in Ca2+ transport. Human calbindin D28k was cloned into a Pet3a vector and expressed in Escherichia coli. The protein was purified in three steps: (i) heat precipitation of bacterial proteins, (ii) ion-exchange chromatography on a DEAE-cellulose column in the presence of calcium, and (iii) ion-exchange chromatography on a DEAE-Sephacel column in the presence of EDTA. The protein was then supplemented with calcium and dialyzed against neutral water. The final yield was 20-50 mg of pure, homogeneous calcium-loaded calbindin D28k per liter of bacterial culture. The identity and purity of the protein were confirmed by immunoblotting, SDS-polyacrylamide gel electrophoresis, and agarose gel electrophoresis in the absence and presence of calcium and 1H NMR spectroscopy. The entire expression and purification protocol takes only 3 days and is easy to scale up and down. It was designed to minimize degradation and deamidation.     

A novel cDNA extension procedure. Isolation of chicken fatty acid synthase cDNA clones

We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.     

The effects of Ca(2+) binding on the conformation of calbindin D(28K): a nuclear magnetic resonance and microelectrospray mass spectrometry study

Calbindin D(28K) is a six-EF-hand calcium-binding protein found in the brain, peripheral nervous system, kidney, and intestine. There is a paucity of information on the effects of calcium binding on calbindin D(28K) structure. To further examine the mechanism and structural consequences of calcium binding to calbindin D(28K) we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D(28K). The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i). apo-calbindin D(28K) has an ordered structure which changes to a notably different ordered conformation upon Ca(2+) loading, (ii). calcium binding is a sequential process and not a simultaneous event, and (iii). EF-hands 1, 3, 4, and 5 take up Ca(2+), whereas EF-hands 2 and 6 do not. Our results support the opinion that calbindin D(28K) has characteristics of both a calcium sensor and a buffer.     

Organization of calbindin D28K-immunoreactive neurons in the dog superior colliculus

We localized calbindin D28K-immunoreactive (IR) neurons in the superior colliculus (SC) of the dog and studied the distribution and effect of enucleation on the distribution of this protein. We also compared this labeling to that of GABA. Calbindin D28K was localized with antibody immunocytochemistry. Calbindin D28K-IR neurons formed three laminar tiers in the SC, one within the lower superficial gray layer (SGL), the second within the upper intermediate gray layers (IGL), and the third within the deep gray layer (DGL). The third tier was not very distinctive when compared with the other two tiers. Calbindin D28K-IR neurons in the SC varied dramatically in morphology and size, and included round/oval, vertical fusiform, stellate, pyriform, and horizontal neurons. Neurons with varicose dendrite were also labeled in the IGL. Enucleation appeared to have no effect on the distribution of calbindin D28K-IR neurons in the contralateral SC. Two-color immunofluorescence revealed that a small percentage (11.20%) of calbindin D28K-IR neurons co-localized with GABA. The current results demonstrate that the patterned distribution of calbindin D28K-IR neurons in the intermediate and deep SC is comparable with other animals, but that the distribution of this protein in the superficial SC is strikingly different from that in previously studied animals. The results also suggest that retinal projection may not control the activity of the expression of calbindin D28K in the dog SC. These results will not only provide valuable knowledge of the basic neurochemical architecture of the dog visual system, but also provide clues for the understanding of the similarities and differences among species.     

Molecular cloning and the complete nucleotide sequence of the creatine kinase-M cDNA from chicken.

The nucleotide sequence of creatine kinase-M (CK-M) cDNA clones has been determined. It includes the entire coding region of 381 amino acids in addition to 5' and 3' untranslated regions. A comparison with a partial sequence from rat CK-M reveals 84% nucleotide sequence homology in the coding region but divergence in the 3' untranslated region. The amino acid sequence is 94% conserved between chicken and rat. Hybridization to RNA immobilized on filters indicates homology between the CK-M 3' untranslated region and additional muscle specific RNA species. The coding region hybridizes only to CK-M RNA.     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.