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.     

Primary structure of human lymphotoxin derived from 1788 lymphoblastoid cell line

The amino acid sequence of human lymphotoxin derived from a 1788 lymphoblastoid cell line was determined. Peptide fragments obtained by trypsin, lysine-C peptidase, cyanogen bromide, and acetic acid cleavage of the intact protein were purified by reverse-phase high performance liquid chromatography and analyzed by amino acid composition and by automated Edman degradation. The protein is 171 amino acids long with a molecular weight of 18,664. It contains one asparagine-linked glycosylation site and lacks cysteine. The salient features of the amino acid sequence of lymphotoxin are described.     

Complete amino acid sequence of human transforming growth factor type beta 2

The complete amino acid sequence of human type beta 2 transforming growth factor (hTGF-beta 2) was determined by automated Edman degradation of S-pyridylethylated hTGF-beta 2 and selected fragments. Cleavage of hTGF-beta 2 by enzymatic and chemical techniques established all the fragments in an unambiguous sequence. Human TGF-beta 2 consists of two disulfide-linked, identical subunits. Each hTGF-beta 2 subunit is a single-chain polypeptide of 112 residues, with a calculated molecular weight of 12,720. Human TGF-beta 2 displays 71.4% sequence homology with the functionally related human TGF-beta 1, and is distantly related (23-40% amino acid identity) to porcine inhibins and activins, the carboxyl-terminal regions of human Müllerian inhibiting substance, and the putative decapentaplegic gene complex protein of Drosophila.     

Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem repeats

The amino acid sequence of human plasma prekallikrein was determined by a combination of automated Edman degradation and cDNA sequencing techniques. Human plasma prekallikrein was fragmented with cyanogen bromide, and 13 homogeneous peptides were isolated and sequenced. Cyanogen bromide peptides containing carbohydrate were further digested with trypsin, and the peptides containing carbohydrate were isolated and sequenced. Five asparagine-linked carbohydrate attachment sites were identified. The sequence determined by Edman degradation was aligned with the amino acid sequence predicted from cDNAs isolated from a lambda gt11 expression library. This library contained cDNA inserts prepared from human liver poly(A) RNA. Analysis of the cDNA indicated that human plasma prekallikrein is synthesized as a precursor with a signal peptide of 19 amino acids. The mature form of the protein that circulates in blood is a single-chain polypeptide of 619 amino acids. Plasma prekallikrein is converted to plasma kallikrein by factor XIIa by the cleavage of an internal Arg-Ile bond. Plasma kallikrein is composed of a heavy chain (371 amino acids) and a light chain (248 amino acids), and these 2 chains are held together by a disulfide bond. The heavy chain of plasma kallikrein originates from the amino-terminal end of the zymogen and is composed of 4 tandem repeats that are 90 or 91 amino acid residues in length. These repeat sequences are also homologous to those in human factor XI. The light chain of plasma kallikrein contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.     

The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain

The complete sequence of 157 amino acids of the light (A) chain of high molecular mass urokinase from human urine was determined. The fragmentation strategy included cyanogen bromide cleavage of the S-carboxymethylated A chain at the methionine and/or tryptophan residues and use of the specific endoproteinase Lys-C. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. C-terminal amino acids of the A chain were determined by consecutive treatment with carboxypeptidase A and B. The amino acid sequence obtained revealed a significant homology to peptide chains of other serine proteinases. Accordingly, the sequence of the A chain can be divided into three domains: 1) The growth factor domain with homologies to murine epidermal growth factor and a particular sequence of bovine clotting factor X, 2) The "kringle" domain with homologies to "kringle" structures, e.g. in plasminogen, and 3) the connecting peptide domain containing the A1 chain of low molecular mass urokinase. Together with the amino acid sequence of the B chain, which was presented by us in an earlier communication, the sequence data presented complete the primary structure of high molecular mass urokinase from human urine.     

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.     

The complete amino acid sequence of low molecular mass urokinase from human urine

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.     

Amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor)

The amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor) was determined by automated Edman degradation using the peptides produced by chemical and enzymatic cleavages of intact factor XII and alpha-factor XIIa. Combining this sequence with the previously determined sequence of beta-factor XIIa (Fujikawa, K., and McMullen, B. A. (1983) J. Biol. Chem. 258, 10924-10933), the complete amino acid sequence of human factor XII has been established. The heavy chain of alpha-factor XIIa is composed of 353 amino acid residues containing one Asn-linked and six probable O-linked carbohydrate chains. The heavy chain of alpha-factor XIIa appears to contain four different domains including a "kringle," a "growth factor" domain, and the "type I" and "type II" domains of fibronectin. The domain organization of factor XII is analogous to those of several fibrinolytic proteins, including tissue plasminogen activator and urokinase, suggesting that factor XII belongs to the same protease subfamily as these two proteins.     

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine lutropin beta-subunit

The amino acid sequence was determined for equine lutropin beta (eLH beta). Large fragments were derived from reduced, carboxymethylated eLH beta by digestion with Staphylococcus aureus V8 protease, by cyanogen bromide cleavage, and by cleavage of acid-labile Asp-Pro bonds. The fragments were purified by gel filtration and high performance liquid chromatography (HPLC). The fragments were sequenced by automated Edman degradation to establish the primary structure of eLH beta. Some peptides were further digested with chymotrypsin and the resulting peptides purified by HPLC. In addition to sequencing by automated Edman degradation, these were also sequenced by the complementary 5-dimethylaminonaphthalene-1-sulfonyl-Edman procedure which enabled us to directly identify glycosylated amino acids. The eLH beta subunit is a glycoprotein of 149 amino acids containing both N- and O-linked oligosaccharides. It possesses a COOH-terminal extension similar to that seen in human chorionic gonadotropin. Carboxypeptidase Y digestions suggest that the COOH terminus is blocked by glycosylation. Interestingly, the amino acid sequence of eLH beta is identical to that of equine chorionic gonadotropin beta (Sugino, H., Bousfield, G. R., Moore, W. T., and Ward, D. N. (1987)J. Biol. Chem. 262, 8603-8609).     

Effect of the cobalt-N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin

The binding of several corrinoids to the binding site of human intrinsic factor, transcobalamin or haptocorrin was investigated, p-Cresolyl cobamide and 2-amino-vitamin B12 are complete corrinoids, whose nucleotide at the lower face of the corrin ring is not coordinated to the cobalt. These corrinoids were greater than or equal to 10(3) times less efficiently recognized by intrinsic factor or transcobalamin than vitamin B12, which contains a Co-coordinated nucleotide. Pseudovitamin B12, with a weak Co-N coordination bond, revealed only moderate affinity to intrinsic factor. From these findings it is concluded that the cobamide binding to intrinsic factor and transcobalamin is strongly affected by the Co-N coordination bonds of their lower cobalt nucleotide ligands. We suggest that the Co-N coordination bond positions the nucleotide at a critical distance to the corrin ring, which is recognized by the binding proteins. Human haptocorrin, however, disclosed to distinctive selectivity regarding the different corrinoid structures. The protein bound all corrinoids with similar efficiency, independent of the strength of their Co-N coordinations, or the structures of their lower Co alpha ligands. Hence, the corrin ring, rather than a structural feature induced by the Co-N coordination, has to be considered responsible for the corrinoid binding to haptocorrin.     

Use of o-phthalaldehyde to reduce background during automated Edman degradation

Background generated during the cleavage step of the Edman degradation is a major obstacle to extended automated amino acid sequencing. It was demonstrated recently by A. S. Bhown, J. C. Bennett, P. H. Morgan, and J. E. Mole (1981, Anal. Biochem. 112, 158-162) that introduction of fluorescamine into the spinning cup reduced background by blocking primary amino groups at cycles where a proline residue is at the exposed amino terminus. A convenient blocking reaction program using o-phthalaldehyde which can be intercalated into the sequencing format of an automated Beckman liquid-phase sequencer is presented. The advantages of o-phthalaldehyde in blocking of primary amines when proline is amino terminal arise from its stability in aqueous solution and the ease of programmed metering of delivery to the sequencer spinning cup. The blocking reaction proved successful not only in extending sequence analyses but also in the elimination of unwanted sequences in selected peptide mixtures without the necessity of purification of the target peptide.     

The amino-acid sequence of beta-lactoglobulin II from horse colostrum (Equus caballus, Perissodactyla): beta-lactoglobulins are retinol-binding proteins

beta-Lactoglobulin isolated from horse colostrum is heterogeneous and contains two components: beta-lactoglobulin I and beta-lactoglobulin II. These two proteins are monomeric and show differences in their electrophoretic mobilities, chain lengths and primary structures. The complete amino-acid sequence of beta-lactoglobulin II was determined by automated Edman degradation of the intact protein and of the peptides derived from these by digestion with trypsin or chymotrypsin and by chemical cleavage with cyanogen bromide. Unlike other beta-lactoglobulins which contain 162 amino acids, horse beta-lactoglobulin II is unique in that it contains 166 amino acids. The additional four amino acids represent an insertion between positions 116 and 117 of other beta-lactoglobulins so far sequenced, including horse beta-lactoglobulin I. Sequence comparison of beta-lactoglobulins I and II from horse colostrum reveals 48 amino acid substitutions (30%). Such a diversity between members of the beta-lactoglobulin gene family has not been encountered before. Sequence comparison with bovine beta-lactoglobulin A shows 85 amino acid replacements accounting for 53% of the residues. The structural homology with human retinol-binding protein may reveal similar biological functions and clues to the origin of milk proteins.     

Primary structure of PDC-109, a major protein constituent of bovine seminal plasma

The two major protein components of bovine seminal plasma, PDC-109 and BSP I, have been purified by gel filtration, partition chromatography and reverse-phase high performance liquid chromatography from an 86% ethanol precipitate of bovine seminal plasma ejaculate. The complete 109-residue amino acid sequence of PDC-109 has been established by automated Edman degradation of the intact peptide as well as its proteolytic digestion and cyanogen bromide cleavage fragments. The 12,774 dalton structure has two structurally similar domains of 38 and 41 amino acids, each containing two disulfide bonds.     

The complete primary structure of a proline-rich phosphoprotein from human saliva.

The complete amino acid sequence of a calcium-binding "proline-rich phosphoprotein," named Protein A, from human saliva was determined by automated and manual Edman degradation of peptides obtained by enzymatic and chemical cleavage of the intact protein. The NH2-terminal pyrrolidone carboxylic acid was identified by means of NMR. The protein consists of 106 amino acids, including 24 residues of proline. The NH2-terminal 32 residues contain 13 of the 15 negatively charged residues including 2 phosphoserines, but only 1 proline. In spite of a high concentration of proline in the COOH-terminal part of the molecule, the longest oligoproline sequence is tetraproline. The protein contains a number of repeated sequences and there are also several sequences of 3 or 4 residues identical with known sequences of collagen, but the characteristic occurrence of glycine in every third position in collagen is not found in salivary Protein A.     

Amino acid sequence of rubber elongation factor protein associated with rubber particles in Hevea latex

The amino acid sequence of rubber elongation factor, a recently discovered protein tightly bound to rubber particles isolated from the commercial rubber tree Hevea brasiliensis, is presented. The role of this protein in rubber elongation and its interaction with prenyltransferase and rubber particles have been discussed in the preceding paper in this series (Dennis, M. S., and Light, D. R. (1989) J. Biol. Chem. 264, 18608-18617). Trypsin, Staphylococcus protease, chymotrypsin, acetic acid, and hydroxylamine cleavage were used to generate peptide fragments that were isolated by reverse phase high pressure liquid chromatography and analyzed by amino acid composition and automated Edman degradation. Each digest contained one blocked peptide identified as the amino terminus. The blocked amino-terminal peptide from the tryptic digest was analyzed by amino acid composition, fast atom bombardment mass spectrometry (molecular ion 1659.9), subdigested with Staphylococcus protease for partial sequence analysis, and finally deblocked with bovine liver acyl-peptide hydrolase removing an acetylalanine to allow analysis by Edman degradation. Rubber elongation factor is 137 amino acids long, has a molecular mass of 14,600 daltons, and lacks four amino acids: cysteine, methionine, histidine, and tryptophan. The NH2 terminus is highly charged and contains only acidic residues (5 of the first 12 amino acids). The first four amino acids are highly represented in other known NH2-terminally acetylated proteins. Comparison of the sequence of rubber elongation factor with other known sequences does not reveal significant sequence similarities that would suggest an evolutionary relationship.     

The primary structure of rat liver ribosomal protein L39

The covalent structure of the rat liver 60 S ribosomal subunit protein L39 was determined. Fourteen tryptic peptides were purified, and the sequence of each was established by a micromanual procedure; they accounted for all 50 residues of L39. The sequence of the NH2-terminal 32 residues of L39, obtained by automated Edman degradation of the intact protein, provided the alignment of the first seven tryptic peptides. Two peptides, CNI (28 residues) and CNII (22 residues), were produced by cleavage of protein L39 with cyanogen bromide and the sequence of CNII was determined by automated Edman degradation. This sequence established the order of tryptic peptides T8 through T14. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. Protein L39 contains 50 amino acids and has a molecular weight of 7308. There are indications that a portion of rat L39 is related to a fragment of Escherichia coli ribosomal protein S1.     

Complete amino acid sequence of human phosphoglycerate kinase. Isolation and amino acid sequence of tryptic peptides

As a part of the elucidation of the complete amino acid sequence of human phosphoglycerate kinase, 46 tryptic peptides, ranging in length from 1 to 26 residues, were isolated and characterized from the reduced and S-carboxymethylated enzyme. The isolated peptides were subjected to sequence analysis by the modified dansyl-Edman degradation procedure and automated Edman degradation technique. The results, together with the data on cyanogen bromide peptides and two additional tryptic peptides from cyanogen bromide peptides reported in the accompanying paper, established the complete amino acid sequence of human erythrocyte phosphoglycerate kinase.     

Pig beta-lactoglobulin I (Sus scrofa domestica, Artiodactyla). The primary structure of the major component

beta-Lactoglobulins from pooled milk (Sus scrofa domestica) are isolated and characterized. The complete primary structure of the major beta-lactoglobulin component I is presented. The amino-acid sequence was elucidated by automated Edman degradation of tryptic peptides and cyanogen bromide cleavage products in a liquid phase sequencer. The tryptic and cyanogen bromide peptides were separated by reverse-phase (RP-2) or size exclusion (TSK 2000 SW) high performance liquid chromatography. Pig beta-lactoglobulin is composed of only 159 amino acids in contrast to other beta-lactoglobulins which contain 162 or 166 amino acids. Sequence alignment with previously sequenced beta-lactoglobulins was obtained by introducing two gaps at positions 115 and 151-152. Thus bovine beta-lactoglobulin A reveals 62 amino-acid substitutions. The phylogenetic distance from horse beta-lactoglobulin I and II is indicated by 49.4% and 62% amino-acid exchanges, respectively. Pig beta-lactoglobulin is a mixture of two chains with Gln or Thr at position 119. The free thiol group is localized at position 59. The structural and functional aspects of beta-lactoglobulins and its role in vitamin A (retinol) transport are discussed.     

Primary structure of the amino-terminal (vitamin K-dependent) region of human prothrombin

The amino acid sequence of residues 1–51 of human prothrombin fragment 1 has been determined. This 51 residue peptide is 1 residue shorter than the comparable bovine region and 8 of the amino acid residues are different. The positions of 10 glutamic acid residues are identical to the γ-carboxylated ones in the bovine species. From this homology and additional evidence, these residues in human prothrombin are considered to be γ-carboxylated. The sequence was completed by automated Edman degradation of the reduced, alkylated fragment and its subfragmentation with trypsin, thermolysin and acid hydrolysis.