Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

Amino acid sequence of chicken liver cathepsin L

The complete amino acid sequences of the heavy and light chains of chicken liver cathepsin L have been determined by automated gas-phase Edman degradation. The heavy and light chains contained 176 and 42 amino acid residues respectively. A glucosamine-based oligosaccharide group was attached to Asn-109 of the heavy chain. Chicken liver cathepsin L had high sequence homology with rat cathepsin H, but exhibited less similarity with rat cathepsin B. Comparisons of cathepsin L with plant cysteine proteinases, such as papain, actinidin and aleurain, reveal high degree of homology.     

The N-terminal amino acid sequences of the heavy and light chains of human cathepsin L. Relationship to a cDNA clone for a major cysteine proteinase from a mouse macrophage cell line.

Human liver cathepsin L consists of a heavy chain and a light chain with Mr values of 25,000 and 5000 respectively. The chains have been purified and their N-terminal amino acid sequences have been determined. The 40 amino acids determined from the heavy chain and 42 amino acids sequenced in the light chain are homologous with the N-terminal and C-terminal regions respectively of the superfamily of cysteine proteinases. Therefore it is likely that the two chains of cathepsin L are derived by proteolysis of a single polypeptide precursor. Of the amino acids sequenced, 81% are identical with the homologous portions of a protein sequence for a major cysteine proteinase predicted from a cDNA clone from a mouse macrophage cell line. This is the closest relative amongst the known sequences in the superfamily and strongly indicates that the protein encoded by this mRNA is cathepsin L. The mouse protein is also probably the major excreted protein of a transformed cell line [Gal & Gottesman (1986) Biochem. Biophys. Res. Commun. 139, 156-162]. The heavy chain is identical in only 71% of its residues with the sequence of ox cathepsin S, providing further evidence that this latter enzyme is probably not a species variant of cathepsin L. The relationship with a second unidentified cathepsin cDNA clone from a bovine library is much weaker (41% identity), and so this clone remains unidentified.     

Amino acid sequence of human liver cathepsin B

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252-residue sequence was obtained by automated solid-phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single-chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.     

Isolation and sequencing of a cDNA clone encoding rat liver lysosomal cathepsin D and the structure of three forms of mature enzymes

We isolated and sequenced a cDNA clone corresponding to the entire coding sequence of rat liver lysosomal cathepsin D. The deduced amino acid sequence revealed that cathepsin D consists of 407 amino acid residues (Mr 44,608) and the 20 NH2-terminal residues seem to constitute a cleavable signal peptide after which 44 amino acid residues follow as a propeptide. Two putative N-linked glycosylation sites and aspartic acid in the active site are as well conserved as those of human lysosomal cathepsin D. In the NH2-terminal sequence analysis of two isolated heavy chains of the mature enzyme, the termini were assigned as tryptophan (118th residue) and glycine (165th or 166th residue), respectively, hence demonstrates that the two heavy chains derive from a split of the single chain of cathepsin D at position between 117th and 118th or between 164th and 165th or 165th and 166th amino acids. We conclude that cathepsin D in rat liver lysosomes is a mixture of three forms composed of a single and two two-chain forms. However, the amounts of the two two-chain forms are low compared with that of the single chain form. Densidometric determination after SDS-PAGE revealed that the two two-chain forms account for less than 5% of the single chain form. There is a 82% similarity in amino acid level between rat and human liver lysosomal cathepsin D.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.     

Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor

Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing.     

Structures at the proteolytic processing region of cathepsin D

The amino acid sequences at the "proteolytic processing regions" of cathepsin Ds have been determined for the enzymes from cows, pigs, and rats in order to deduce the sites of cleavage as well as the function of the proteolytic processing of cathepsin D. For bovine cathepsin D, the "processing region" sequence was determined from a peptide isolated from the single-chain enzyme. The COOH-terminal sequence of the light chain and the NH2-terminal sequence of the heavy chain were also determined. The processing region sequence of porcine cathepsin D was determined from its cDNA structure, and the same structure from rat cathepsin D was determined from the peptide sequence of the single-chain rat enzyme. From sequence homology to other aspartic proteases whose x-ray crystallographic structures are known, such as pepsinogen and penicillopepsin, it is clear that the processing regions are insertions to form an extended beta-hairpin loop between residues 91 and 92 (porcine pepsin numbers). However, the sizes of the processing regions of cathepsin Ds from different species are considerably different. For the enzymes from rats, cows, pigs, and human, the sizes of the processing regions are 6, 9, 9, and 11 amino acid residues, respectively. The amino acid sequences within the processing regions are considerably different. In addition, the proteolytic processing sites were found to be completely different in the bovine and porcine cathepsin Ds. While in the porcine enzyme, an Asn-Ser bond and a Gly-Val bond are cleaved to release 5 residues as a consequence of the processing; in the bovine enzyme, two Ser-Ser bonds are cleaved to release 2 serine residues. These findings would argue that the in vivo proteolytic processing of the cathepsin D single chain is probably not carried out by a specific "processing protease." Model building of the cathepsin D processing region conformation was conducted utilizing the homology between procathepsin D and porcine pepsinogen. The beta-hairpin structure of the processing region was found to (i) interact with the activation peptide of the procathepsin D in a beta-structure and (ii) place the Cys residue in the processing region within disulfide linkage distance to Cys-27 of cathepsin D light chain. These observations support the view that the processing region of cathepsin D may function to stabilize the conformation of procathepsin D and may play a role in its activation.     

Bovine factor VII. Its purification and complete amino acid sequence

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).     

Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.     

Amino-terminal amino acid sequence and heterogeneity in glycosylation of rat renal renin

We isolated 7.4 mg of pure renin from 2 kg of rat kidneys using affinity chromatography on pepstatin-aminohexyl-Sepharose and an octapeptide renin inhibitor, H-77-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that renin consists of two polypeptide chains linked by a disulfide bond, one of Mr = 36,000 (heavy chain) and the other of Mr = 3,000 (light chain). The amino-terminal 10-amino acid sequences of the heavy and the light chains were identical to the sequences beginning at Ser72 and Asp355, respectively, of the amino acid sequence of preprorenin deduced from the renin cDNA sequence. Amino acid sequencing of the carboxyl-terminal peptide of the heavy chain, generated by digestion with lysyl endopeptidase, showed that the carboxyl-terminal residue of the heavy chain is Phe. Thus, the propeptide of prorenin is cleaved after Thr71, followed by removal of two amino acids, Arg353 and Asn354, the result being formation of the heavy and light chains. Thus, the site of cleavage of rat prorenin is after a nonbasic amino acid, in contrast to the cleavage of the propeptide after a pair of basic amino acids in mouse submaxillary renin, human renal renin, and many secretory proteins. Treatment of renin with neuraminidase or glycopeptidase F had no apparent effect on the charge heterogeneity of renin. Glycosylation probably does not contribute to charge heterogeneity.     

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver arginase

Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.     

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.     

Amino acid sequence of rat kidney gamma-glutamylcysteine synthetase

gamma-Glutamylcysteine synthetase catalyzes the first step in the synthesis of glutathione. The enzyme isolated from rat kidney has two subunits (heavy, Mr 73,000; and light, Mr 27,700) which may be dissociated by treatment with dithiothreitol. The heavy subunit exhibits all of the catalytic activity of the isolated enzyme and also feedback inhibition by glutathione. The light subunit has no known function and may not be an integral part of the enzyme. cDNA clones encoding rat kidney gamma-glutamylcysteine synthetase were isolated from a lambda gt11 cDNA library by immunoscreening with antibody against the isolated enzyme and further screening with oligonucleotide probes derived from several peptides whose sequences were determined by the Edman method. The nucleotide sequence of the mRNA for the heavy subunit was deduced from the sequences of the cDNA of three such clones. The sequence, which codes for 637 residues (Mr 72,614), contains all four of the independently determined peptide sequences (approximately 100 residues). This amino acid sequence shows extremely low overall similarity to that of gamma-glutamylcysteine synthetase isolated from Escherichia coli.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].     

Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.     

Purification and amino acid sequence of chicken liver cathepsin L

Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.     

Subunit structures of three human myeloperoxidases

The heavy and the light subunits of human myeloperoxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) I, II, and III were isolated from the reduced and S-carboxymethylated enzymes. These three enzymes have the same terminal amino acid sequences and similar chemical compositions in both subunits. The NH2-terminal sequences of the heavy and light subunits were determined to be Val-Asn-Cys-Glu-Thr- and Thr-Cys-Pro-Glu-Gln-, respectively; a heterogeneity was observed in the NH2-termini of the latter subunits for the three enzymes. As for COOH-termini, the sequences -(Asn, 2 Leu, Ala, Ser, Trp)-Arg-Glu-Ala and -Ala-Arg were obtained for the heavy and the light subunits, respectively. The heavy subunits contained 8-10 mol/mol of glucosamine. On the basis of these results and the amino acid sequence deduced from cDNA clones, the heavy subunits probably correspond to amino acids 279-744 and the light subunits to amino acids (164-167)-272. For the heavy subunits, Ser-745, which was predicted as the COOH-terminal amino acid from the nucleotide sequence, was removed. The light subunits were also processed at their COOH-termini by 6 residues. Four or five high mannose type carbohydrate chains were attached to the heavy subunits.     

Conservation in rat liver of light and heavy subunit sequences of mammalian ferritin. Presence of unique octopeptide in the light subunit

Ferritin, an iron-storage protein found in all life forms examined, is composed of varying proportions of two subunits of different molecular weight, heavy (H) and light (L). Using cDNA clones, we have determined the nucleotide sequence corresponding to the mRNA of the L-subunit of rat liver ferritin. The coding region of 546 nucleotides (182 amino acids) is flanked by 5'- and 3' -untranslated regions of approximately 130 and 150 nucleotides, respectively. The rat liver L-subunit amino acid sequence derived from the reading frame of the cDNA showed 88% and 82% homology, respectively, with the amino acid sequences of horse spleen ferritin (Heusterspreute, M., and Crichton, R. R. (1981) FEBS Lett. 129, 322-327), and human spleen ferritin (Wustefeld, C., and Crichton, R. R. (1982) FEBS Lett. 150, 43-48), thus demonstrating evolutionary conservation of the L-subunit sequence. However, a major difference between the rat and the horse and human sequences is the insertion of an octopeptide near the COOH-terminus of the rat protein resulting in a slightly longer peptide chain in this species. The reading frame and parts of the derived amino acid sequence including the octopeptide sequence were confirmed by direct amino acid sequencing of cyanogen bromide peptides from rat liver ferritin. Minor fragments of rat liver ferritin, presumably derived from the H-subunit, were also isolated after cyanogen bromide treatment. On sequencing, these H-peptides showed limited homology with regions of the L-sequence but extensive homology with published H-sequences from human liver and spleen. The H-subunit sequence did not contain the octopeptide found as part of the L-subunit sequence.