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.     

Molecular cloning of cDNA for human complement component C1s. The complete amino acid sequence

The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s.     

The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

Protein chemical characterization of Mucor pusillus aspartic proteinase. Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment

The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.     

Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri

The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid-soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues. The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.     

Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster.

We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.     

Localization of thrombin cleavage sites in the amino-terminal region of bovine protein S

Protein S is a vitamin K-dependent plasma protein. It functions as a cofactor to activated protein C in the inactivation of factors Va and VIIIa by limited proteolysis. Protein S is very sensitive to proteolysis by thrombin which reduces its calcium ion binding and leads to a loss of its cofactor activity. We have now determined the sequence of the 100 amino-terminal amino acid residues and localized the thrombin cleavage sites. Protein S contains 11 gamma-carboxyglutamic acid residues in the amino-terminal region (residues 1-36). This part of protein S is highly homologous to the corresponding parts in the other vitamin K-dependent clotting factors, whereas the region between residues 45 and 75 is not at all homologous to the other clotting factors. Thrombin cleaves two peptide bonds in this part of protein S, first at arginine 70 and then at arginine 52. The peptide containing residues 53-70 is released from protein S after thrombin cleavage. The amino-terminal fragment, residues 1-52, is linked to the large carboxyl-terminal fragment by a disulfide bond, which involves cysteine 47. After residue 78, protein S is again homologous to factors IX and X and to proteins C and Z, but not to prothrombin. Position 95 is occupied by a beta-hydroxyaspartic acid residue.     

The primary structure of bovine cellular retinoic acid-binding protein

The complete amino acid sequence of bovine adrenal gland cellular retinoic acid-binding protein (CRABP) has been determined. The primary structure was established by analyses of cyanogen bromide fragments and peptides obtained by trypsin and Staphylococcus aureus protease digestions. The polypeptide chain of bovine CRABP comprises 136 amino acid residues. From partial sequence information, CRABP has been shown to be homologous to cellular retinol-binding protein, myelin protein P2, and the fatty acid-binding Z-protein. A comparison of the complete amino acid sequences of the members of this protein family, which also includes the rat intestinal fatty acid-binding protein, shows that CRABP is more similar to cellular retinol-binding protein and protein P2 than to the fatty acid-binding proteins. All five proteins are very similar in their NH2-terminal regions, suggesting that this part is important for a property common to the members of this protein family. This is the first report of a complete amino acid sequence of a CRABP.     

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.     

Sites of methyl esterification on the aspartate receptor involved in bacterial chemotaxis

The methyl esterification of the aspartate receptor involved in chemotaxis has been studied in order to clarify the role of receptor modification. Receptors were methyl esterified in an in vitro system using S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Methyl esterified receptors were digested with trypsin and radioactive tryptic peptides were purified using high performance liquid chromatography. Comparing the amino acid composition of the modified peptides with the DNA sequence of the receptor gene, two regions of the polypeptide chain which contain methyl esterified residues were identified. The regions are homologous and contain a strongly conserved 13 amino acid sequence. One region, containing up to three modified residues, is near the middle of the protein; the other, containing one modified residue, is near the carboxyl terminus.     

Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A

The amino acid sequence of subunit A of the potato chymotryptic inhibitor I was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide, N-bromosuccinimide and dilute acid, and by digestion with trypsin, thermolysin, pepsin and papain. The molecule consists of a single polypeptide chain of 84 residues, which contains two homologous regions each of 13 amino acids. The protein does not appear to be homologous with any other known proteinase inhibitors.     

Extensive sequence homology between IgA receptor and M proteins in Streptococcus pyogenes

Many strains of Streptococcus pyogenes are known to express a receptor for IgA. The complete nucleotide sequence of the gene for such a receptor, protein Arp4, has been determined. The deduced amino acid sequence of 386 residues includes a signal sequence of 41 amino acids and a putative membrane anchor region, both of which are homologous to similar regions in other streptococcal surface proteins. The processed form of the IgA receptor has a length of 345 amino acids and a calculated molecular weight of 39544. The N-terminal sequence of the processed form is different from that previously found for a similar IgA receptor isolated from a S. pyogenes strain of type M60. The sequence of protein Arp4 shows extensive homology to the C-terminal half of streptococcal M proteins, but not to the streptococcal IgG receptor protein G or staphlyococcal protein A. Apart from the membrane anchor, this homology includes a sequence of 119 amino acid residues containing three repeated units and a 54-residue sequence without repeats. The protein expressed in Escherichia coli is found in the periplasmic space, in which it constitutes the major protein. Protein Arp4 is the first example of a surface protein that has both immunoglobulin-binding capacity and structural features characteristic of M proteins.     

Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase.

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.     

Amino acid sequence of human protein Z, a vitamin K-dependent plasma glycoprotein

Protein Z is a vitamin K-dependent glycoprotein isolated and characterized from human and bovine plasma. A cDNA coding for human protein Z has been obtained by the isolation of phage clones from a liver cDNA library and in vitro amplification of two other liver libraries. Protein Z is synthesized with a prepro-leader sequence of 40 amino acids. The mature protein is composed of 360 residues including a Gla domain of 13 carboxyglutamic acid residues, two epidermal growth factor domains, and a carboxyl terminal region which is highly homologous to the catalytic domain of serine proteases. Human protein Z, however, contains an Asp instead of Ser and a Lys instead of His in the catalytic triad of the active site.     

Amino acid sequence of P-57, a neurospecific calmodulin-binding protein

The amino acid sequence was determined for bovine brain P-57, a neurospecific, membrane-associated, calmodulin-binding protein. It consists of a single 239-residue polypeptide chain blocked at its amino terminus and containing an unusually hydrophilic amino acid composition. Seventy percent of the molecule is composed of Glu/Gln, Ala, Lys, Asp/Asn, and Pro; there is only one aromatic residue. A lack of favorable cleavage sites required that a particularly wide variety of digests and subdigests be performed to obtain appropriate sets of overlapping peptides. This protein is clearly homologous with the cDNA-derived sequence of mouse brain P-57, although the bovine protein is 12 amino acid residues longer; the homology is less obvious in the middle sections of the two sequences. Bovine brain P-57 lacks homology with any other protein in an updated sequence database. A segment reported to interact with calmodulin (Arg-Gly-His-Ile-Thr-Arg-Lys-Lys-Leu) is placed at residues 43-51 within the only extended segment of P-57 that carries the net positive charge that would favor that interaction. There is no hydrophobic segment characteristic of many proteins that interact with membranes.     

Sequence homology and immunologic cross-reactivity of human cytomegalovirus with HLA-DR beta chain: a means for graft rejection and immunosuppression

A peptide (Leu-Gly-Arg-Pro-Asp-Glu-Asp-Ser-Ser-Ser-Ser-Ser-Ser-Ser-Cys) that was identical to residues 82 through 96 of a predicted protein of 208 amino acids from the immediate-early region (IE-2) nucleic acid sequence of human cytomegalovirus was chemically synthesized. By computer analysis, the first five amino acids of this peptide showed sequence homology to the beta chain of the human histocompatibility complex HLA-DR. The homologous amino acids, 53 through 57, were located in a region that is conserved between the human DR beta chain and the beta chain of the H-2 class II histocompatibility antigen for mice. The shared region between the IE-2 protein and DR beta chain were similar in both hydrophilicity and predicted beta-turn potential. The IE-2 viral peptide induced antibodies that specifically recognized the human DR beta chain. These observations describe a protein encoded by the IE-2 region of human cytomegalovirus that contains sequence homology and shows immunologic cross-reactivity with a conserved domain of HLA-DR and suggest a mechanism to explain how human cytomegalovirus infection contributes to graft rejection after transplantation.     

The amino acid sequence of Phaseolus aureus L. (mung-bean) cytochrome c

The amino acid sequence of Phaseolus aureus L. (mung-bean) cytochrome c has been determined. The molecule consists of a single polypeptide chain of 111 amino acid residues and is homologous with other mitochondrial cytochromes c. Comparison with the amino acid sequence of wheat-germ cytochrome c (Stevens, Glazer & Smith, 1967) shows 14 differences. On alignment with mammalian cytochromes c, mung-bean cytochrome c has an N-acetylated ;tail' of eight amino acid residues similar to that found in wheat-germ cytochrome c. Of the 22 positions in wheat-germ cytochrome c that contain amino acid residues unique to these positions, 20 were found to contain the same ones in mung-bean cytochrome c. The in-N-trimethyl-lysine residues reported for wheat-germ cytochrome c (Delange, Glazer & Smith, 1969) in positions 72 and 86 were also found in these positions in mung-bean cytochrome c. The sequence was determined from 3mumol, by using chymotryptic and tryptic peptides which were analysed by the ;dansyl'-Edman method (Gray & Hartley, 1963a), with confirmation by amino acid analysis.     

Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses

Murine leukemia viruses contain a low molecular weight basic protein, designated p10, which binds to single-stranded nucleic acids. The complete amino acid sequence of p10 from the Rauscher strain of virus has been determined. The partial amino acid sequences of p10s from Moloney, Friend, AKR, Gross, radiation leukemia, and BALB/2 viral strains have also been determined using microsequencing techniques. Rauscher p10 is composed of 56 amino acid residues; the other p10s are similar in size but differ from Rauscher by a few conservative amino acid substitutions. The structure of Rauscher p10 was compared to the structure of a functionally homologous protein from Rous avian sarcoma virus. The comparison revealed regions of amino acid sequence homologies which indicate a phylogenetic relationship between the murine and avian viral strains. The analyses revealed a periodic placement of three Cys residues and a Gly-His sequence. A structure involving these residues is found once in the murine protein and twice in the avian protein. A similar structure is seen in the single stranded nucleic acid binding protein of bacteriophage T4. However, in the latter case, the order of amino acid residues is inverted.     

Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis

The amino acid sequence of a protease from the crayfish Astacus fluviatilis has been determined from overlapping sets of peptides derived largely by cleavage at Met, Lys, or Arg residues. The protein comprises 200 amino acid residues in a single polypeptide chain, corresponding to a molecular mass of 22,614 daltons. Two disulfide bonds link Cys-42 to Cys-198 and Cys-64 to Cys-84. The sequence of this invertebrate protease appears to be unique since it has no homologous relationship to any of the known protein sequences.