Complete amino acid sequence of the catalytic chain of human complement subcomponent C1-r

The amino acid sequence of human C1-r b chain hs been determined, from sequence analysis performed on fragments obtained by CNBr cleavage, dilute acid hydrolysis, tryptic cleavage of the succinylated protein, and subcleavages by staphylococcal protease. The polypeptide chain contains 242 amino acids (Mr 27 096), and the sequence shows strong homology with other mammalian serine proteases. The histidine, aspartic acid, and serine residues of the active site (His-57, Asp-102, and Ser-195 in bovine chymotrypsinogen) are located at positions 39, 94, and 191, respectively. The chain which lacks the "histidine-loop" disulfide bridge, contains five half-cystine residues, of which four (positions 157-176 and 187-217) are homologous to residues involved in disulfide bonds generally conserved in serine proteases, whereas the half-cystine residue at position 114 is likely to be involved in the single disulfide bridge connecting the catalytic b chain to the n-terminal a chain. Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 51 and 118.     

Amino acid sequence of a lectin from Japanese frog (Rana japonica) eggs

The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented. The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide. The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus. Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111. The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin. They are also homologous to human angiogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases.     

三叶肽：从结构到功能

三叶结构域是一段由38－39个氨基酸组成的多肽序列,其中包含6个高度保守的半胱氨酸残基,这6个半胱氨酸残基以1－5,2－4,3－6的交联方式形成三对二硫,窝囊鑫肽链折叠成特征性的三叶结构。已发现的哺乳动物三叶肽有三种：pS1、SP及ITF。三叶肽通常位于消化道腔面的粘膜层,具有保护和修复功能,在维持粘膜的完整性中发挥着重要作用。     

Identification of the active site residues of Pseudomonas aeruginosa protease IV. Importance of enzyme activity in autoprocessing and activation

Protease IV is a lysine-specific endoprotease produced by Pseudomonas aeruginosa whose activity has been correlated with corneal virulence. Comparison of the protease IV amino acid sequence to other bacterial proteases suggested that amino acids His-72, Asp-122, and Ser-198 could form a catalytic triad that is critical for protease IV activity. To test this possibility, site-directed mutations by alanine substitution were introduced into six selected residues including the predicted triad and identical residues located close to the triad. Mutations at any of the amino acids of the predicted catalytic triad or Ser-197 caused a loss of enzymatic activity and absence of the mature form of protease IV. In contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form. A purified proenzyme that accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P. aeruginosa. Furthermore, similarities of protease IV to the lysine-specific endoprotease of Achromobacter lyticus suggested three possible disulfide bonds in protease IV. These results identify the catalytic triad of protease IV, demonstrate that autodigestion is essential for the processing of protease IV into a mature protease, and predict sites essential to enzyme conformation.     

The complete primary structure of phospholipase A2 from human pancreas

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.     

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 and location of the disulfide bonds in bovine beta 2 glycoprotein I: the presence of five Sushi domains.

beta 2 glycoprotein I is a plasma protein with the ability to bind with various kinds of negatively charged substances. The complete amino acid sequence and the location of all the disulfide bonds of bovine beta 2 glycoprotein I were determined. Bovine beta 2 glycoprotein I consists of 326 amino acid residues with five asparagine-linked carbohydrate chains. Homology with the human protein was calculated to be 83%. Eleven disulfide bonds in bovine beta 2 glycoprotein I constitute four characteristic domains, Sushi domains, and one modified form of a Sushi domain.     

Crystal structure of carboxylase reaction-oriented ribulose 1, 5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1. 39) obtained from a thermophilic red alga Galdieria partita has the highest specificity factor of 238 among the Rubiscos hitherto reported. Crystal structure of activated Rubisco from G. partita complexed with the reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) has been determined at 2.4-A resolution. Compared with other Rubiscos, different amino residues bring the structural differences in active site, which are marked around the binding sites of P-2 phosphate of 2-CABP. Especially, side chains of His-327 and Arg-295 show the significant differences from those of spinach Rubisco. Moreover, the side chains of Asn-123 and His-294 which are reported to bind the substrate, ribulose 1,5-bisphosphate, form hydrogen bonds characteristic of Galdieria Rubisco. Small subunits of Galdieria Rubisco have more than 30 extra amino acid residues on the C terminus, which make up a hairpin-loop structure to form many interactions with the neighboring small subunits. When the structures of Galdieria and spinach Rubiscos are superimposed, the hairpin region of the neighboring small subunit in Galdieria enzyme and apical portion of insertion residues 52-63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other.     

Amino acid sequence of rat mast cell protease I (chymase)

The amino acid sequence has been determined for rat mast cell protease I (RMCP I), a product of peritoneal mast cells. The active enzyme contains 227 residues, including three corresponding to the catalytic triad characteristic of serine protease (His-57, Asp-102, and Ser-195 in chymotrypsin). A computer search for homology indicates 73% and 33% sequence identity of RMCP I with rat mast cell protease II from mucosal mast cells and bovine chymotrypsin A, respectively. When the structure of RMCP I is compared to those of cathepsin G from human neutrophils and two proteases expressed in activated lymphocytes, 48-49% of the sequences are identical in each case. RMCP I has six half-cystine residues at the same positions as in RMCP II, cathepsin G, and the two lymphocyte proteases, suggesting disulfide pairs identical with those reported for RMCP II. A disulfide bond near the active site seryl residue and substrate binding site, present in pancreatic and plasma serine proteases, is not found in RMCP I or in the other cellular proteases. These results indicate that RMCP I and other chymotrypsin-like proteases of granulocyte and lymphocyte origin are more closely related to each other than to the pancreatic or plasma serine proteases.     

The amino acid sequence of Limulus C-reactive protein. Evidence of polymorphism

The amino acid sequence, the positions of the disulfide bonds, and the site of glycosylation for the three subunits of Limulus C-reactive proteins (CRPs) 1.1, 1.4, and 3.3 have been established. The three subunits were shown to exist approximately in equimolar amount and are tightly associated. The hexagonal structure of Limulus CRP, as revealed by electron microscopic studies of Fernandez-Moran et al. (Fernandez-Moran, H., Marchalonis, J., and Edelman, G. M. (1968) J. Mol. Biol. 32, 467-469) might consist of two each of the subunits. The three subunits share an identical amino-terminal sequence of 44 residues and a carboxyl-terminal sequence from residues 206 to 218. Microheterogeneity exists to the extent of 10 to 11% for the entire protein. The positions of 6 half-cystines that form the three disulfide bonds and the site of glycosylation are constant in all subunits. Sequence analyses of peptides derived from enzymatic and chemical cleavages of affinity purified Limulus CRP indicate that subunits other than the three mentioned above exist in the hemolymph. Limulus CRP is therefore polymorphic. Topological analyses of Limulus CRPs, human CRP, rabbit CRP, human amyloid P-component, and Syrian hamster female protein indicate that the seven proteins may originate from the same ancestral gene. Using the topological data generated from the amino acid sequences of the proteins, we calculate that human and Limulus CRPs diverged about 500 million years ago. This figure is in general agreement with the evolutionary distance postulated by anthropological estimation of 400-500 million years.     

Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement

The amino acid sequence of one of the major lysosomal membrane glycoproteins, lysosome-associated membrane protein 1 (lamp-1), was deduced from its cDNA sequence (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928). This amino acid sequence suggests that lamp-1 contains a hinge-like structure and could form disulfide bridges that are observed in the immunoglobulin superfamily. To test this possibility, we have determined the positions of the disulfide bridges by isolating and sequencing cystine-containing peptides which contain disulfide bridges. The results indicate that disulfide arrangement of lamp-1 is different from that of immunoglobulins. Each molecule contains, in total, four loops formed by disulfide bonds, and each loop contains 36-39 amino acid residues. However, none of the disulfide bonds connects two domains that are separated by a hinge-like structure. The results indicate that the hinge region has no ordered structure, and the relative positions of the two domains can be altered in space. Examination of the ultrastructure of lamp-1 by electron microscopy showed that the hinge-like structure actually functions as a hinge. These results indicate that the lamp-1 molecule represents a novel family of glycoproteins with unique structural properties.     

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.     

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.     

Molecular cloning of the E1 beta subunit of the rat branched chain alpha-ketoacid dehydrogenase.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.     

The primary structure of cassowary (Casuarius casuarius) goose type lysozyme

The complete amino acid sequence of cassowary (Casuarius casuarius) goose type lysozyme was analyzed by direct protein sequencing of peptides obtained by cleavage with trypsin, V8 protease, chymotrypsin, lysyl endopeptidase, and cyanogen bromide. The N-terminal residue of the enzyme was deduced to be a pyroglutamate group by analysis with a LC/MS/MS system equipped with the oMALDI ionization source, and then confirmed by a glutamate aminopeptidase enzyme. The blocked N-terminal is the first reported in this enzyme group. The positions of disulfide bonds in this enzyme were chemically identified as Cys4-Cys60 and Cys18-Cys29. Cassowary lysozyme was proved to consist of 185 amino acid residues and had a molecular mass of 20408 Da calculated from the amino acid sequence. The amino acid sequence of cassowary lysozyme compared to that of reported G-type lysozymes had identities of 90%, 83%, and 81%, for ostrich, goose, and black swan lysozymes, respectively. The amino acid substitutions at PyroGlu1, Glu19, Gly40, Asp82, Thr102, Thr156, and Asn167 were newly detected in this enzyme group. The substituted amino acids that might contribute to substrate binding were found at subsite B (Asn122Ser, Phe123Met). The amino acid sequences that formed three alpha-helices and three beta-sheets were completely conserved. The disulfide bond locations and catalytic amino acid were also strictly conserved. The conservation of the three alpha-helices structures and the location of disulfide bonds were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.     

Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II.

The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII (C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.     

Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis

The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identification of similar molecules in a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site-directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the presence of reactive histidine residues within the active site. Site directed mutagenesis suggested an essential role in catalysis for two histidine residues (His-136 and His-272), which are conserved in all sequences. Mutations of two additional histidines (His-138 and His-151), conserved only in eukaryotes, resulted in reduced neutral sphingomyelinase activity. In addition to sphingomyelin, the enzyme also hydrolyzed lysophosphatidylcholine. Exposure to an oxidizing environment or modification of cysteine residues using several specific compounds also inactivated the enzyme. Site-directed mutagenesis of eight cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvement of cysteines in the formation/breakage of disulfide bonds, which could underlie the reversible inactivation by the oxidizing compounds. Cellular localization studies of a series of deletion mutants, expressed as green fluorescent protein fusion proteins, demonstrated that the transmembrane region contains determinants for the endoplasmic reticulum localization.     

Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli

To determine the modes of three disulfide linkages in the heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli, we synthesized STh(6-18), which consists of 13 amino acid residues and has the same intramolecular disulfide linkages as native STh [(1985) FEBS Lett. 181, 138-142], by stepwise and selective formation of disulfide bonds using different types of removable protecting groups for the Cys residues. Synthesis of the peptide with different modes of disulfide bond formation provided three peptides consistent with standard STh(6-18) in their physicochemical and biological properties, thereby indicating that the disulfide bonds in STh(6-18) are Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys.     

Genes encoding alpha-heavy chains of rabbit IgA: characterization of cDNA encoding IgA-g subclass alpha-chains

cDNA molecules encoding rabbit IgA alpha-heavy chains have been synthesized and six of these have been characterized. The complete nucleotide sequence of one cDNA, p 19 (942bp), showed that it encoded all but the N-terminal 57 amino acid residues of the constant region of alpha-chains. The cDNA molecules were subcloned into the expression vector pUC8 and E. coli were transformed. Radioimmunoassay of the molecules synthesized by these clones showed that all six cDNA molecules encoded alpha-chains of the IgA-g subclass. Comparison of the amino acids encoded by the alpha-cDNA with the amino acid sequence of mouse and human alpha-chains showed that although all of the intradomain disulfide bonds appear to be conserved, some positions, probably involved in interchain disulfide bonds, are not conserved. We propose that secretory component is covalently bound to cysteine 299 and/or cysteine 301 of the CH2 domain of mouse and human alpha-chains. The results from Southern blot analysis of genomic DNA with 32P-cDNA suggests that the rabbit genome has multiple C alpha genes.