Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.     

Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator.

1. One of the activation products of C4, C4b, was prepared, and the reactive thiol group on the alpha'-chain was radioactively labelled with iodo[2-14C]acetic acid. The alpha'-chain was isolated and the N-terminal amino acid sequence of the first 13 residues was determined. 2. C4b was cleaved by C3bINA in the presence of C4b-binding protein and C4d and C4c isolated. The radioactive label and therefore the reactive thiol group were located to C4d. 3. C4c was reduced and alkylated and the two alpha'-chain fragments of C4c were separated. 3. The molecular weights, amino acid analyses and carbohydrate content of the three alpha'-chain fragments were determined. C4d has a mol.wt. of 44500 and a carbohydrate content of 6%. The two alpha'-chain fragments of C4c have mol.wts. of 25000 (alpha 3) and 12000 (alpha 4) and carbohydrate contents of 10 and 22% respectively. 4. The N-terminal amino acid sequences of C4d, the alpha 3 and the alpha 4 fragments were determined for 18, 24 and 11 residues respectively and, by comparison with the N-terminal sequence of the C4b alpha'-chain, the 25000-mol.wt. fragment (alpha 3) was shown to be derived from the N-terminal part of the alpha'-chain. 5. C-Terminal analyses were done on the alpha'-chain and its three fragments. Arginine was found to be the C-terminal residue of C4d and of the alpha 3 fragment. The C-terminal residue of the alpha'-chain and of the alpha 4 fragment could not be identified. The order of the three fragments of the alpha'-chain is therefore: alpha 3(25000)--C4d(44500)--alpha 4(12000). The specificity of C3bINA is for an Arg--Xaa peptide bond.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

The structure of a naturally occurring 10K polypeptide derived from the amino terminus of bovine thyroglobulin

A combination of data derived from peptide sequencing and nucleic acid sequencing of cloned cDNA fragments has been used to define the complete amino acid sequence of a 10,000 M.W., thyroxine containing polypeptide derived from bovine thyroglobulin. This fragment, TG-F, which was obtained following reduction and alkylation, has been placed at the amino terminus of the parent protein with hormone located at residue 5 in the primary sequence of the thyroglobulin molecule. The carboxyl terminal sequence of this fragment -Cys-Gln-Leu-Gln is found on the N-terminal side of a lys residue, suggesting that the peptide bond cleavage which occurs to produce this 80 residue fragment from the parent (330K) thyroglobulin chain is a gln-lys. In addition, the amino acid sequence of this 10K fragment contains: No sequence which would be a substrate for glycosylation and no carbohydrate. Several repeated homologous amino acid sequences. A striking number of beta-bends predicted from Chou-Fasman analyses, particularly near its carboxyl terminus.     

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.     

On the primary structure of human plasminogen and plasmin. Purification and characterization of cyanogen-bromide fragments.

Most of the cyanogen bromide fragments obtained from human plasminogen and plasmin have been purified using combinations of gel filtration and ion-exchange chromatography. The purified fragments have been characterized by molecular weight determination (dodecyl sulphate electrophoresis), amino acid analysis, carbohydrate analysis and direct NH2-terminal amino acid sequence determination. Since some of the purified fragments were compounds with uncompletely cleaved methionyl bonds it was possible to clarify the organization of most of the cyanogen bromide fragments in the plasminogen molecule. The fragment containing the arginyl-valyl bond cleaved during the second step of the activation process is further identified. It is also shown that the microheterogeneity that normally exists in human plasminogen probably has its origin in several sites. One such site is situated in the light (B) chain of plasmin, while another is situated in the carboxyterminal part of the heavy (A) chain. Neither of these sites seems to contain sialic acid.     

An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.     

The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C.

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.     

Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules.

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.     

Amino-terminal sequence, synthesis, and membrane insertion of glycoprotein B of herpes simplex virus type 1.

Glycoprotein B (gB) was purified from cells infected with two strains (KOS and F) of herpes simplex virus type 1. Determination of amino acid sequence at the NH2 termini revealed, by comparison with amino acid sequence deduced from previously published nucleotide sequence, that gB is made with a cleavable signal sequence of 29 or 30 amino acids, depending on the virus strain. Analysis of gB translated in vitro in the presence and absence of membranes showed that gB is inserted into membranes and glycosylated cotranslationally; a large portion of the gB polypeptide made in vitro is protected from proteolysis by membranes; the large protected fragment carries N-linked carbohydrate and is probably the NH2 terminus based on locations of signals for the addition of N-linked carbohydrate; and the size of the protected fragment is 93 kilodaltons (kDa) for gB made in vitro and associated with dog pancreas membranes, whereas both 93- and 98-kDa protected fragments can be detected for gB made in vivo. These last results are consistent with a previous proposal that gB may traverse the membrane three times.     

The covalent structure of hepatic microsomal epoxide hydrolase. I. Isolation and characterization of the cyanogen bromide fragments

Microsomal epoxide hydrolase was purified to homogeneity from phenobarbital-induced rabbit liver for the purpose of determining the complete amino acid sequence. All of the expected 11 cyanogen bromide fragments of epoxide hydrolase were isolated by a combination of gel filtration and high pressure liquid chromatography. Each was characterized by its amino acid composition and NH2-terminal amino acid sequence. The complete amino acid sequences of the eight small fragments, from 5-29 residues, were determined.     

The amino acid sequence of the light chain of human high-molecular-mass kininogen

The complete amino acid sequence of the light chain of human high-molecular-mass kininogen has been determined. The peptide chain contains 255 amino acid residues. The half-cystine, which forms the disulfide bridge to the heavy chain, was identified in position 225. Nine carbohydrate attachment sites were found. All carbohydrate side chains are O-glycosidically linked. Alignment of the present sequence with the bovine kininogen light chain sequence shows a high degree of homology, except for an extension of 22 amino acids within the histidine-rich part of the sequence. The histidine-rich region may have arisen by gene multiplication during evolution.     

Periodate oxidation studies of human pituitary follicle-stimulating hormone

A preparation of human pituitary follicle-stimulating hormone was subjected to periodate oxidation, borohydride reduction and acid hydrolysis. Comparison of the analysis of the remaining intact carbohydrate and amino acid units with the analyses of the original material and identification of the carbohydrate fragments permit some structural assignments to the molecule of follicle-stimulating hormone. The results of radioimmunological assay of fragments of the molecule of follicle-stimulating hormone suggest that, although the carbohydrate component is essential for biological activity, it is not a requirement for immunological activity, which appears to be a function of the protein moiety.     

Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin.

The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.     

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.     

The amino-acid sequence of kangaroo pancreatic ribonuclease

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.     

The carbohydrate moiety of Japanese monkey pepsinogens. Its composition and site of attachment to protein.

Identification and determination of the carbohydrate component of Japanese monkey pepsinogens have been performed, and the amino acid sequence around the carbohydrate chain has been investigated. Glycopeptides were prepared by successive digestion of pepsinogens with thermolysin and aminopeptidases. Analyses of their carbohydrate composition by paper and gas-liquid chromatography showed the presence of 4 glucosamine, 6 galactose, 6–8 mannose, and 8–10 fucose residues per molecule of the carbohydrate chain, among which the high content of fucose is especially unique. The amino acid sequence of a major glycopeptide was deduced to be Ile-Gly-Ile-Gly-Thr-Pro-Gln-Ala-Asn, in which the asparagine residue is the site of attachment of the carbohydrate chain.     

Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin

The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 263, 11536-11539) was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor.     

Comparison of the amino acid sequences of hen plasma-, yolk-, and white-riboflavin binding proteins

The amino acid sequence of hen egg yolk-riboflavin binding protein (yolk-RBP) was determined by conventional methods. The sequence was identical with that of hen egg white-riboflavin binding protein except that their carboxyltermini were different, that of yolk-RBP lacked 11 or 13 amino acid residues, while hen plasma-RBP had the same C-terminal sequence as white-RBP. This indicated that the C-terminal 11 or 13 amino acid residues in plasma-RBP might be cleaved off during the incorporation from the blood into the oocyte or in the yolk fluid. Yolk-RBP had the same characteristics as white-RBP, such as N-terminal pyroglutamic acid, polymorphism in the amino acid sequence (Lys/Asn) at the fourteenth residue from the N-terminal end, carbohydrate chains attached to both Asn(36) and Asn(147) residues, and phosphate groups bound to some serine residues in the sequence of Ser(185) to Ser(197) as a cluster. These results led us to the conclusion that yolk- and white-RBPs are bio-synthesized from the same gene in the different organs (liver and oviduct). The carbohydrate composition of yolk-RBP was identical to that of plasma-RBP but different from that of white-RBP showing that the processing of the carbohydrate chains in the liver was different from that in the oviduct.     

The amino acid sequences of erabutoxins, neurotoxic proteins of sea-snake (Laticauda semifasciata) venom

1. Erabutoxin b was reduced, S-carboxymethylated and hydrolysed with trypsin. Seven tryptic fragments were isolated by column chromatography and paper electrophoresis. Some of the fragments were further hydrolysed with alpha-chymotrypsin, pepsin, Nagarse, Proctase A or Proctase B. The amino acid sequences of the fragment peptides were determined by subtractive Edman degradation. 2. From the tryptic digest of reduced, S-carboxymethylated and trifluoroacetylated erabutoxin b two fragments were isolated. From the amino acid composition of the fragments and from the terminal sequence studies on the reduced and S-carboxymethylated erabutoxin b, the sequence of the above seven tryptic fragments was elucidated. 3. The tryptic digestion of reduced and S-carboxymethylated erabutoxin a gave fragments, only one of which was different from the corresponding fragment from erabutoxin b. The amino acid sequence analysis of the fragment peptide showed that the only difference between erabutoxins a and b was that the former had asparagine and the latter had histidine at position 26.