Internal quadruplication in the structure of human interstitial retinol-binding protein deduced from its cloned cDNA

Interstitial retinol-binding protein (IRBP) is a glycoprotein that shuttles retinoids between the retina and pigment epithelium and is secreted by the photoreceptor cells of the vertebrate eye. Human retina cDNA libraries in lambda gt10 were screened with a previously isolated human IRBP probe (H.4 IRBP), yielding five overlapping cDNA clones generating a 4223-base sequence. A 17-kilobase pair clone (HGL.3) isolated by screening a human genomic library in EMBL3 with H.4 IRBP yielded a 2.5-kilobase pair SstI fragment that overlapped the 5' end of the cDNA sequence by 329 nucleotide residues. An open reading frame encoded the N-terminal sequence of human IRBP and predicted a protein consisting of 1262 amino acids with a molecular mass of 136,600. Two putative N-linked glycosylation sites were identified. The translated sequence suggests that there is a 16-amino acid presumptive signal peptide rich in hydrophobic residues and with a high alpha-helix probability preceding the N terminus of the mature protein. The amino acid sequence of human IRBP could be aligned with 87% identity with the amino acid sequences of 31 peptides (605 residues) purified from a tryptic digest of bovine IRBP. The protein sequence of human IRBP contains four duplicated segments (302-310 residues in length) with 33-38% identity. From the degree of identity between the bovine and human sequences, it is possible that IRBP evolved by several gene duplications that occurred 600-800 million years ago, before the emergence of the vertebrates.     

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.     

Cytosolic epoxide hydrolase from liver of control and clofibrate-treated mice. Structural comparison by HPLC peptide mapping

Cytosolic epoxide hydrolases purified from livers of control and clofibrate-induced male C57B1/6 mice were compared. The proteins were reduced, alkylated and cleaved with trypsin and chymotrypsin. The digests were analyzed by HPLC and no qualitative differences were observed in the peptide mapping profiles of the two types of epoxide hydrolase preparation. The amino acid compositions and N-terminal residues of selected tryptic peptides also gave identical results for the control and clofibrate-induced mice. Both intact proteins have e-amino-blocked N-termini. The two enzyme forms are concluded to have highly similar, if not identical, primary structures.Abbreviations HPLC high-performance liquid chromatography - DABITC dimethylaminoazobenzene isothiocyanate     

A reinvestigation of the amino acid sequences of bovine, rabbit, monkey, and human myelin basic proteins

In order to determine whether bovine, rabbit, and monkey myelin basic proteins (BPs) have the sequence Gly-His or His-Gly at positions corresponding to bovine sequence 76-77, we isolated the tryptic peptides encompassing the sequence in question in these proteins and cleaved them into dipeptides with dipeptidyl aminopeptidase I (EC 3.4.14.1). Analysis by gas chromatography/mass spectrometry of the dipeptides released showed that in no case did His follow Gly or Gly precede His. The identification of peptides Ala-Gln and His-Gly (bovine BP) and Ser-His and Gly-Arg (rabbit and monkey BPs) established the His-Gly sequence. A similar sequence analysis of tryptic peptide (80-91) of human BP confirmed the sequence Thr-Gln-Asp-Glu-Asn-Pro (80-85).     

Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone

We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.     

The expression of multiple forms of troponin T in chicken-fast-skeletal muscle may result from differential splicing of a single gene

Troponin T isolated from chicken fast skeletal muscle has been shown to be present in three different molecular forms, one in breast and two in leg muscle. The three forms differ in both size and charge. Troponin T from breast muscle has a molecular mass of 33.5 kDa and a pI of about 7. Of the two leg muscle forms the larger has a molecular mass of 30.5 kDa and a pI of about 8.5 and the smaller a molecular mass of 29.8 kDa and a pI of about 10. Considerably more heterogeneity has been found in the leg than in the breast muscle proteins although this is not reflected in their N-terminal sequences. The reason for this is not clear. Troponin T from breast or leg muscle can be phosphorylated with troponin T kinase at the single serine residue at the N-terminus. No difference in the rate or extent of phosphorylation could be found between proteins from breast or leg muscle. The three proteins have been shown to differ only in the amino acid sequence of their N-terminal tryptic peptides. These peptides are of different length, that from breast troponin T being 58 residues and those from leg troponin T being 36 and 42 residues, these differences account for the difference in molecular mass of the parent proteins. Despite this difference the sequence of the first 12 and last 14 residues is identical in all three N-terminal peptides. The remainder of the sequence of the smallest peptide is also repeated in the other two but they each contain an extra piece of unique sequence. On the basis of these sequences it is proposed that chicken troponin T is coded for by a single gene containing, at the 5' end, a number of small exons and that three different mRNA molecules may be produced by alternative pathways of RNA splicing. The possible significance of these N-terminal sequence variations is discussed.     

The primary structure of human dopamine-beta-hydroxylase: insights into the relationship between the soluble and the membrane-bound forms of the enzyme.

A full length dopamine-beta-hydroxylase (DBH) cDNA clone was isolated from a human pheochromocytoma lambda gt11 library. Both structural and functional evidence confirms the authenticity of the clone: (i) antibodies selected with fusion proteins generated by positive clones precipitate DBH activity, (ii) the sequence of three internal DBH tryptic peptides are included in the deduced DBH sequence, (iii) the previously reported N-terminal 15 amino acids of bovine DBH exhibits a nearly complete identity with that predicted for human DBH. The polypeptide chain of DBH comprises 578 amino acids corresponding to an unmodified protein of 64 862 daltons and is preceded by a cleaved signal peptide of 25 residues. DBH exists in both membrane-bound and soluble forms. The hydropathy plot reveals no obvious hydrophobic segment, except the signal peptide. S1 mapping analysis indicates no diversity in the 5' and 3' extremities of the DBH mRNA. Taken together with available biochemical data, these observations suggest that the membrane attachment of DBH probably results from a post-translational modification, glypiation being the most likely candidate. Comparative amino acid sequence analysis establishes that DBH shares no homology with the other catecholamine synthesizing enzymes, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase.     

Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.     

Amino Acid Sequence of Porcine Myelin Basic Protein

The myelin basic protein (BP) of pig brain was cleaved into its constituent tryptic peptides and the amino acid composition of each was determined. Those tryptic peptides that had not been sequenced previously were cleaved with dipeptidyl peptidases and the resulting dipeptides were trimethylsilated, separated by gas chromatography, and identified by mass spectrometry. Carboxypeptidases B and Y were used to establish the COOH-terminal sequences of some of the tryptic peptides; one tryptic peptide (sequence 76-92) was cleaved with thermolysin and the thermolytic peptides were analyzed. From the results of the present study together with those reported previously, it has been possible to determine the complete amino acid sequence of the protein. The protein consists of 172 residues and has a theoretical molecular weight of 18,604. Its amino acid sequence is identical with that reported for the homologous bovine protein with the following exceptions: Ser replaces (bovine) Ala2; His-Gly is inserted between Arg9 and Ser10; Ala replaces Ser45; His and Gly replace Gly76 and His77, respectively; Pro replaces Ser131 and Ser135; Ala is inserted between Gly142 and His143; and Gln replaces His143.     

Human pituitary thyrotropin: isolation and chemical characterization of its subunits

The subunits of human pituitary thyrotropin have been separated and purified by countercurrent distribution and exclusion chromatography. The NH₂-terminal sequence of the β subunit is identical to that of the β subunit of bovine thyrotropin. However, amino acid composition and peptide map of tryptic and chymotryptic digests as well as compositions of tryptic and chymotryptic peptides suggest that the amino acid sequence of the α subunit is identical to that of the α subunit of human interstitial cell stimulating hormone.     

Isolation and amino acid sequence analysis of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase.

3 beta-Hydroxysteroid dehydrogenase/steroid isomerase has been purified to homogeneity from bovine adrenal glands. A single protein of molecular weight 42,090 +/- 40 containing both enzyme activities has been isolated. Approximately 86% of the amino acid sequence of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase has been obtained by sequencing peptides isolated from digests with trypsin and lysyl endopeptidase and by chemical cleavage with CNBr. The sequence obtained is identical with that of the deduced amino acid sequence of the bovine ovarian 3 beta-hydroxysteroid dehydrogenase/steroid isomerase [Zhao et al. (1989) FEBS Lett. 259, 153-157], with the exception that the N-terminal methionine residue found in the bovine ovarian sequence is not present in the mature bovine adrenal enzyme. On the basis of the primary structure and comparisons with other NAD+ binding proteins, we propose a structural model of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase localizing the NAD+ binding site as well as the membrane-anchoring segment.     

Origin of the NEFA and Nuc Signal Sequences

The human protein NEFA binds calcium, contains a leucine zipper repeat that does not form a homodimer, and is proposed (along with the homologous Nuc protein) to have a common evolutionary history with an EF-hand ancestor. We have isolated and characterized the N-terminal domain of NEFA that contains a signal sequence inferred from both endoproteinase Asp-N (Asp-N) and tryptic digests. Analysis of this N-terminal sequence shows significant similarity to the conserved multiple domains of the mitochondrial carrier family (MCF) proteins. The leader sequence of Nuc is, however, most similar to the signal sequences of membrane and/or secreted proteins (e.g., mouse insulin-like growth factor receptor). We suggest that the divergent NEFA and Nuc N-terminal sequences may have independent origins and that the common high hydrophobicity governs their targeting to the ER. These results provide insights into signal sequence evolution and the multiple origins of protein targeting. Received: 20 February 1997 / Accepted: 28 July 1997     

Comparative studies on the primary structure of acetylcholinesterases from bovine caudate nucleus and bovine erythrocytes

1. Comparison of partial amino acid sequences of G2-acetylcholinesterase (AChE) from bovine erythrocytes and G4-AChE from bovine caudate nucleus revealed no differences in primary structure between the two enzymes. The first 33 residues of the N-terminal sequences were identical. 2. In addition, the amino acid sequences of four peptides generated by tryptic and cyanogen bromide cleavage were identical for bovine erythrocyte and brain AChE, suggesting one identical major coding exon for the adult bovine AChE forms. Comparison of these sequences with that of fetal bovine serum AChE (Doctor et al., 1988), showed differences in residues 16, 181, 212, and 216. 3. Deglycosylation studies of the two adult enzyme forms revealed that the core protein of erythrocyte AChE has an approximately 4 kDa lower molecular mass than brain AChE. This most probably reflects differences in the C-terminal sequences of the two enzymes.     

Hypomethylation of the interphotoreceptor retinoid-binding protein (IRBP) promotor and first exon is linked to expression of the gene.

The interphotoreceptor retinoid-binding protein (IRBP) is limited in expression to retinal photoreceptor cells and a subset of pinealocytes. We have obtained a genomic clone containing the entire coding region and 7 kb of 5' flanking sequence. As a first step in studying IRBP gene regulation we have examined the CpG methylation patterns of the entire IRBP gene in expressing and non-expressing human cells. This has been done by isolation of high molecular weight DNA from Y-79 cells grown in suspension or attached to poly-D-lysine, which synthesize IRBP at different levels, and from human lymphocytes, which were shown by northern analysis to lack IRBP message. The DNA was digested by either Hpa II, Msp I, or Hha I. Southern blots were prepared with these digests and hybridized with probes made from fragments covering the complete genomic clone. Probes from the first exon, the introns and the 3' end gave banding patterns which showed no differences between the expressing cells and the lymphocytes. A probe from the very 5' end did not give a clear banding pattern, probably due to the presence of repetitive elements in the probe. However, a Hind III probe covering the 5' flanking 3 kb and the beginning of the first exon hybridized with a 1.8 kb band in Hpa II digests of Y-79 cells which was not present in Hpa II digests of lymphocyte DNA. In addition, a 2.1-2.3 kb Hha I band was found only in the Y-79 DNA digests. Sequence analysis of the promoter region indicated that these bands were due to hypomethylation of sites within a CpG rich island from -1578 to -1108 in the promoter and hypomethylation of sites in the beginning of the first exon. A Hha I site between the CpG island and the first exon was not hypomethylated in the expressing Y-79 cells. We propose that hypomethylation of the CpG rich island of the IRBP promoter and the first exon is linked to the expression of this gene.     

Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.

To purify and characterize the isoform of prostaglandin endoperoxide synthase (rPGSi) induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles, solubilized cell extracts were subjected to anionic exchange chromatography, column fractions were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and rPGSi was visualized by immunoblotting and silver staining techniques. Immunoreactive rPGSi and peroxidase activity co-eluted at pH 6.5 and 6.0. Amino-terminal amino acid sequence of three immunoreactive rPGSi bands (Mr = 72,000, 70,000, and 59,000) were identical, Mr = 59,000 being a proteolytic fragment. Alignment of the amino-terminal sequences of rPGSi with those of ovine PGS (oPGS) indicated that only 15 of 26 residues were identical (58% identity). In contrast, rPGSi was closely related to the deduced amino acid sequence of PGS-related cDNA clones isolated from chicken and mouse cell lines, with 77% (20/26 residues) and 96% (25/26 residues) identity, respectively. Whereas tryptic digests of oPGS generated fragments of Mr = 38,000 and Mr = 33,000, only a small peptide appeared cleaved from the carboxyl terminus of rPGSi. Peroxidase activity of partially purified rPGSi exhibited lower apparent Km and maximal velocity than that of oPGS. Collectively, these results document the existence of a novel rat PGS isoform (based on purification, enzymatic activity, and amino-terminal amino acid sequence) which is hormonally induced and obligatory for a known biological process, ovulation.     

Studies on the covalent structure of eland pancreatic ribonuclease.

Studies on the covalent structure of eland (Taurotragus oryx) pancreatic ribonuclease have been performed on tryptic and thermolysin digests. The first 45 residues have been determined with a Beckman sequencer. From the remaining part of the sequence only those peptides were sequenced that differed in amino acid composition with the corresponding peptide of bovine ribonuclease. Eland pancreatic ribonuclease differs in four positions from bovine pancreatic ribonuclease A, but more differences due to a different state of amidation may be present. The absence of an Asn-X-Thr/Ser sequence in the covalent structure of eland ribonuclease (asparagine 34 has been substituted by aspartic acid) explains the absence of a glycosidated component in eland ribonuclease.     

Primary amino acid sequence of bovine dopamine beta-hydroxylase

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.     

N-terminal amino acid sequences of the hemopexins from chicken, rat and rabbit

The N-terminal amino acid sequences of the hemopexins purified from the plasma of rat, rabbit and chicken were compared with each other and with that of human hemopexin. Although the N-terminal sequences differ among these species, residues 2, 3 and 14 are identical in all four hemopexins. Ten of the first 28 residues are identical in all but the chicken protein. When introducing gaps into the sequence, a much greater homology is observed between the human and rat or rabbit hemopexins (60%) than when the sequences were compared directly (40%).     

Isolation and amino acid sequence of bovine platelet factor 4

Bovine platelet factor 4 was isolated by affinity chromatography using dextran sulfate Sepharose and purified by subsequent gel filtration. The complete amino acid sequence of this 88-residue, 9505-Da protein was determined by isolation and analysis of the overlapping peptides from tryptic and Staphylococcus aureus hydrolysates of reduced, carboxymethylated, and reductive methylated protein. Primary structure comparison was made between bovine platelet factor 4, human platelet factor 4, and human beta-thromboglobulin. The bovine platelet factor 4 amino-terminal region, which contains two unique phenylalanine residues, is extended by 15 residues relative to human platelet factor 4. The bovine carboxy-terminal region is extended by three residues relative to human platelet factor 4 and differed from beta-thromboglobulin in the absence of two additional terminal residues. Bovine platelet factor 4 shares sequence similarities proportionately with both human platelet factor 4 and beta-thromboglobulin. The sequences of the lysine-rich carboxy-terminal putative heparin binding domains are essentially identical for all three proteins. The heparin neutralizing potencies of bovine and human platelet factor 4 are similar: 40 USP units of heparin neutralized per milligram protein, as measured by a modified chromogenic substrate assay. Heparin neutralization was lost by reduction of the disulfide bonds, but only attenuated by tryptic digestion of the intact protein.