Partial amino acid sequence of porcine elastase II. Active site and the activation peptide regions

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.     

Potato lectin: a modular protein sharing sequence similarities with the extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors)

Potato (Solanum tuberosum) lectin, is a chimeric chitin-binding protein comprised of a lectin domain fused to a hydroxyproline-rich glycoprotein domain. Here peptide sequence information from both domains is presented. A partial sequence of a major tryptic peptide T2: Leu-Pro-Ser-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-(His)-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp- was similar to the ‘P3’ type extensin major repetitive sequence: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-suggesting common evolutionary origins for the extensins and the hydroxyproline-rich glycoprotein (HRGP) domain of potato lectin. Furthermore, alignment of three chymotryptic peptides from potato lectin, C1: Cys-Gly-Thr-Thr-Ser-Asp-Tyr, C2: Cys-Ser-Pro-Gly-Tyr, and C8: Thr-Gly-Glu-Cys-Cys-Ser-Ile with similar sequences from the hevein lectin family indicates that they have homologous chitin-binding domains, and hence have common evolutionary origins. Finally, all plant chitin-binding domains examined bore a remarkable sequence similarity, particularly in the spacing of Cys residues, to the disintegrins (platelet aggregation inhibitors) which occur in crotalid and viperid snake venoms. As such, sequence similarities not only identify potato lectin as a member of both the hevein and extensin families of plant proteins, but also suggest that an archetypal polypeptide module gave rise to both the plant chitin-binding domain and the reptile disintegrins.     

Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.)

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree (Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC₅₀-values as low as 1 μg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.     

Primary structure of a 21-kD protein from potato tubers

A 21-kD protein isolated earlier from potato tubers (Solanum tuberosum L.) has two isoforms, with pI 6.3 and 5.2, which were separated by fast protein ion-exchange chromatography on a Mono Q column. The primary structures of the two forms consisted of 187 and 186 amino acid residues. Both isoforms are composed of two polypeptide chains, designated A and B, linked by a single disulfide bond between Cys-146 of the A chain and Cys-7 of the B chain. The amino acid sequences of the A chains of the two forms, consisting of 150 residues each, differ in a single amino acid residue at position 52 (Val --> Ile), while the B chains, containing 37 and 36 residues, respectively, have substitutions at nine positions (Leu-8 --> Ser-8, Lys-25--Asp-26 --> Asn-25--Glu-26, Ile-31--Ser-32 --> Val-31--Leu-32, Lys-34--Gln-35--Val-36--Gln-37 --> Gln-34--Glu-35--Val-36). Both isoforms form stable inhibiting complexes with human leukocyte elastase and are less effective against chymotrypsin and trypsin.     

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.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Amino acid sequence of the light chain derived from a rabbit anti-p-azobenzoate antibody of restricted heterogeneity

The amino acid sequence of the light chain from a specifically purified rabbit (No. 2717) anti-p-azobenzoate antibody preparation (b4 allotype) of restricted heterogeneity has been determined. This light chain is composed of 216 residues, including seven half-cystine residues located at positions 23, 80, 88, 134, 171, 194 and 216. Three intrachain disulfide bonds appear to be present in contrast to only two disulfide bonds as has been so far described for Bence Jones protein and light chains of human and mouse. This light chain was sequenced by isolating the tryptic peptides, sequencing the peptides and establishing their order within the molecule. Unambiguous identification of the overlaps was achieved by taking into account the partially characterized tryptic peptides from citraconic anhydride-treated light chains and chymotryptic and peptic peptides from digests of both untreated and citraconylated light chains. Comparison of this amino acid sequence with the amino acid sequence of the car?ylterminal half of the b4 light chains from unimmunized rabbits reveals differences at positions 165, 166, 169 and 176 indicating the existence of more than one sequence in the b4 “constant” region. There is substantial sequence homology between the variable half of 2717 light chain and human Bence Jones protein. Indeed, 46 positions in the V region (42%) are occupied by the same residues in this light chain and in human subgroup V_κIII.     

Amino acid sequence of the monomer subunit of the giant multisubunit hemoglobin from the earthworm Pheretima sieboldi

The giant extracellular hemoglobin of the earthworm Pheretima sieboldi is mainly composed of two heme-containing subunits: a monomer; chain I and a disulfide-bonded trimer of chains II, III and IV. Both subunits can be separated easily by gel filtration under alkaline conditions. The amino acid sequence of chain I has been determined. It is composed of 141 residues, has two half-cystine residues forming a intrachain disulfide bridge, and has a molecular mass of 16911 Da including a heme group. Heterogeneity was found at position 37 (His or Ser). The amino acid sequence of Pheretima chain I showed 30-50% identity with those of eight heme-containing chains of Lumbricus and Tylorrhynchus hemoglobins. The sequences of nine chains of annelid giant hemoglobins were compared separately in the functionally essential central exonic region and structurally essential side exonic regions, and a phylogenetic tree was constructed. The amino acid substitution rate for the central exon was found to be about 1.5 times slower than that for the side exons.     

Primary structure of barwin: a barley seed protein closely related to the C-terminal domain of proteins encoded by wound-induced plant genes.

Barwin is a basic protein with pI above 10 and molecular mass 13.7 kDa isolated from aqueous extracts of barley seed. The complete amino acid sequence of 125 residues has been determined by a combination of conventional protein sequencing, plasma desorption mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Three disulfide bridges have been localized as Cys31-Cys63, Cys52-Cys86, and Cys66-Cys123 both by 1H nuclear magnetic resonance spectroscopy and by plasma desorption mass spectrometry. The N-terminal residue was identified as pyroglutamate. Barwin is closely related to a peptide segment of 122 residues at the C-terminal region of the proteins encoded by two wound-induced genes in potato plants, win1 and win2, and a protein encoded by the hevein gene of rubber tree. In 77 sequence positions of 125 the barwin, win1, win2, and hevein protein sequences have amino acid sequence identity, when two gaps--one of two residues allowing for the insert of Gly23 and Ala24 and one allowing for the insert of Thr97 in the barwin sequence--are introduced in the latter three. The close sequence similarity with the proteins encoded by the wound-induced potato and rubber tree genes and the ability of the protein to bind saccharides suggest that barwin might belong to a group of proteins involved in a common defense mechanism in plants.     

Partial amino acid sequence in the N-terminal region of an anti-pneumococcal immunoglobulin heavy chain of allotype a2 (Short Communication)

The amino acid sequence of the N-terminal 48 residues of the heavy chain derived from a homogeneous rabbit antibody to type III pneumococci is described. This chain of allotype a(2) is compared with other rabbit heavy chains of allotypes a(1), a(2) and a(3). Within the N-terminal 25 positions, two chains which carry the same allotype a(2) possess identical amino acid sequences, but differ markedly from heavy chains of allotypes a(1) and a(3). Sequence variability is observed in residues 26-27 and 30-34, but not in residues 35-48.     

Botulinum type A neurotoxin digested with pepsin yields 132, 97, 72, 45, 42, and 18 kD fragments

Botulinum neurotoxin (NT) serotype A is a dichain protein made of a light and a heavy chain linked by at least one interchain disulfide; based on SDS-polyacrylamide gel electrophoresis their molecular masses appear as 147, 52, and 93 kD, respectively. Digestion of the NT with pepsin under controlledpH (4.3 and 6.0), time (1 and 24 hr), and temperature (25 and 30°C) produced 132, 97, 42, and 18 kD fragments. The three larger fragments were isolated by ionexchange chromatography. The 132 and 97 kD fragments are composed of 52 kD light chain and 72 and 45 kD fragments of the heavy chain, respectively. The sequences of amino terminal residues of these fragments were determined to identify the pepsin cleavage sites in the NT, which based on nucleotide sequence has 1295 amino acid residues (Binzet al., J. Biol. Chem. 265, 9153, 1990). The 42 kD fragment, beginning with residue 866, is the C-terminal half of the heavy chain. The 18 kD fragment, of which the first 72 residues were identified beginning with residue 1147, represents the C-terminal segment of the heavy chain. The 132 kD fragment (residue 1 to 1146) is thus a truncated version of the NT without its 18 kD C-terminal segment. The 97 kD fragment (residue 1 to 865) is also a truncated NT with its 42 kD C-terminal segment excised. These peptic fragments contain one or two of the three functional domains of the NT (binds receptors, forms channels, and intracellularly inhibits exocytosis of the neurotransmitter) that can be used for structure-function studies of the NT. This report also demonstrates for the first time that of the six Cys residues 453, 790, 966, 1059, 1234, and 1279 located in the heavy chain the later four do not form interchain disulfide links with the light chain; however, Cys 1234 and 1279 contained within the 18 kD fragment form intrachain disulfide. The electrophoretic behaviors of type A NT and its fragments in native gels and their comparison with botulinum NT serotypes B and E as well as tetanus NT suggest that each NT forms dimers or other aggregates and the aggregation does not occur when the 42 kD C-terminal half of the heavy chain is excised. Thus, the C-terminal half of the heavy chain appears important in the self-association to form dimers.     

Study of the glycosylation of apolipoprotein H

Apolipoprotein H is a single chain polypeptide composed of 326 amino acids highly glycosylated. Its carbohydrate content is approximately 19% of the molecular weight. We show that it is rich in sialic acid linked alpha (2-6) to galactose or N-acetylgalactosamine. Sialic acid is not alpha (2-3) linked to galactose. Galactose is beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to N-acetylgalactosamine. Carbohydrate O-linked chains (mainly sialic acid) are alpha (2-6) linked to galactose or N-acetylgalactosamine. Galactose is also organised in O-linked chains and beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to acetylgalactosamine. Concanavalin A lectin was used to isolate two groups of apolipoprotein H molecules bearing biantennary and truncated hybrids and high mannose and hybrid oligosaccharides. Apolipoprotein H fails to bind lysine-Sepharose. Our results thus show that it presents truncated hybrid or hybrid-type carbohydrate chains which bear few unmasked mannose residues as a terminal sugar. Biochemical analysis of carbohydrate structures conducted on single isoforms separated through IEF revealed that no specific carbohydrate complex is bound to a single isoform.     

Conversion of α-Terpineol to 8,9-Epoxy-p-menthan-1-ol

The complete amino acid sequence of pokeweed lectin-B (PL-B) has been analyzed by first sequencing seven lysylendopeptidase peptides derived from the reduced and S-pyridylethylated PL-B and then connecting them by analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides linked to Asn96 and Asnl39, and has a molecular mass of 34,493 Da. PL-B is composed of seven repetitive chitin-binding domains having 48–79% sequence homology with each other. Twelve amino acid residues including eight cysteine residues in these domains are absolutely conserved in all other chitin-binding domains of plant lectins and class I chitinases. Also, it was strongly suggested that the extremely high hemagglutinating and mitogenic activities of PL-B may be ascribed to its seven-domain structure.     

Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein

A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s). A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein. The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end. Five potential N-glycosylation sites were found in each of the two chains of factor XI. The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI. Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide. Each repeat probably forms a separate domain containing three internal disulfide bonds. The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases. The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein.     

Co- and post-translational processing of the hevein preproprotein of latex of the rubber tree (Hevea brasiliensis)

Hevein is a chitin-binding protein of 43 amino acids found in the lutoid body-enriched fraction of rubber tree latex. A hevein cDNA clone (HEV1) (Broekaert, W., Lee, H.-i., Kush, A., Nam, C.-H., and Raikhel, N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637) encodes a putative signal sequence of 17 amino acids followed by a polypeptide of 187 amino acids. Interestingly, this polypeptide has two distinct domains: an amino-terminal domain of 43 amino acids, corresponding to mature hevein, and a carboxyl-terminal domain of 144 amino acids. To investigate the mechanisms involved in processing of the protein encoded by HEV1, three domain-specific antisera were raised against fusion proteins harboring the amino-terminal domain (N domain), carboxyl-terminal domain (C domain), and both domains (NC domain). Translocation experiments using an in vitro translation system show that the first 17-amino acid sequence encoded by the cDNA functions as a signal peptide. Immunoblot analysis of proteins extracted from lutoid bodies demonstrates that a 5-kDa protein comigrated with purified mature hevein and cross-reacted with N domain- and NC domain-specific antibodies. A 14-kDa protein was recognized by C domain- and NC domain-specific antibodies. A 20-kDa protein was cross-reactive with all three antibodies. Microsequencing data further suggest that the 5-kDa (amino-terminal domain) and 14-kDa (carboxyl-terminal domain) proteins are post-translational cleavage products of the 20-kDa polypeptide (both domains) which corresponds to the proprotein encoded by HEV1. In addition, it was found that the amino-terminal domain could provide chitin-binding properties to a fusion protein bearing it either amino terminally or carboxyl terminally.     

Potato lectin: an updated model of a unique chimeric plant protein

A complete cDNA encoding a potato tuber lectin has been identified and sequenced. Based on the deduced amino acid sequence, the still enigmatic molecular structure of the classical chimeric potato lectin could eventually be determined. Basically, the potato lectin consists of two nearly identical chitin-binding modules, built up of two in-tandem arrayed hevein domains that are interconnected by an extensin-like domain of approximately 60 amino acid residues. Although this structure confirms the 'canonical' chimeric nature of the Solanaceae lectins, it differs fundamentally from all previously proposed models. The new insights in the structure are also discussed in view of the physiological role of the Solanaceae lectins.     

Structural features of plant chitinases and chitin-binding proteins

Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains, of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now, and are used to describe the structures of the other ones. Conserved positions of Cys residues can be taken as evidence for identically located disulfide bridges or cysteine residues. The current classification of chitinases is unsatisfactory and needs to be replaced by an evolutionarily more correct one. As the currently known three-dimensional structures of chitinases are those from barley and the rubber tree, Hevea brasiliensis, it is proposed to adopt the designation b-type (classes I, II and IV) and h-type (classes III and V) chitinases, respectively.     

Determination of amino acid sequences of two subunits in sarcosine oxidase from Corynebacterium sp. U-96

The primary structures of the C and D subunits of sarcosine oxidase from Corynebacterium sp. U-96 were determined by sequencing the peptide fragments derived from their enzymatic digestions. The C and D subunits were shown to be composed of 199 and 92 residues, respectively. Each amino acid sequence showed a high homology with the sequence of the corresponding subunit from Corynebacterium sp. P-1. However, there were some differences between these two species, that is, four N-terminal residues were truncated in the C subunit, but six C-terminal residues were truncated in the D subunit. The D subunit contained three cysteine residues, but no disulfide bonds are in the subunit. Overall sequences of both subunit showed no homology with any other protein in the data base.     

Heterogeneity of the covalent structure of the blue copper protein umecyanin from horseradish roots

The covalent structure of umecyanin has been determined by a combination of classical Edman degradation sequence analysis and plasma desorption, laser desorption, and electrospray ionization mass spectrometry. The preparation appeared to contain two isoforms having either a valine (75%) or an isoleucine (25%) residue at position 48. The polypeptide chain of 115 amino acids is strongly heterogeneous at its C-terminal end as a result of proteolytic cleavages at several places within the last 10 residues. The major fraction of the umecyanin preparation is only 106 residues long. The C-terminal tail 107–115 contains mainly alanine and glycine residues and a single hydroxyproline residue. In the native protein there is a disulfide bridge between Cys 91 and Cys 57, but in the apoprotein there is a disulfide shift that involves Cys 91 and one of the four copper binding residues (Cys 85). The three other ligand binding residues are His 44, His 90, and Gin 95. This tetrad of amino acids is the same as occurs in other type 1 copper proteins from plants such as cucumber peeling cupredoxin and lacquer tree stellacyanin. The umecyanin isoforms are glycoproteins with a glycan core having the same carbohydrate composition as that of horseradish peroxidase, a fact that is convincingly supported thanks to the high accuracy of the electrospray mass spectrometric technique. We suggest that the glycan may play a role in the association of the protein to the cellular membrane, but the precise functional role of umecyanin remains to be determined. We also discuss the evolutionary position of umecyanin in relation to the type 1 copper proteins in general.