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

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

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.     

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.     

Structural relationship of an apolipoprotein (a) phenotype (570 kDa) to plasminogen: homologous kringle domains are linked by carbohydrate-rich regions

At least six allelic forms of apolipoprotein(a), differing in molecular mass, could be detected by immunoblot analysis. One of these phenotypes with a molecular mass of 570 kDa has been investigated. After reduction and carboxymethylation it was digested with trypsin and the resulting peptides were separated by gel filtration and reverse phase HPLC. The tryptic fragments sequenced comprised a total of 356 amino acids. The N-terminus of apo(a) was highly homologous to the start of the kringle 4 domain from human plasminogen and the majority of the tryptic peptides isolated was also homologous to sequences from this kringle. At least five homologous "kringle 4" domains are present in apolipoprotein(a) whereby one domain occurs more frequently than the others. A carbohydrate-rich peptide was also obtained in high yield. This glycopeptide connects two "kringle 4" domains and contains one N-glycoside within the kringle and six potential O-glycosides in the linking region. From the recovery it can be estimated that this peptide occurs several times within the whole apolipoprotein (a) sequence. The high carbohydrate content is in sharp contrast to that of human plasminogen. Other peptides sequenced indicate that apo (a) also contains domains homologous to the kringle 5 and protease regions of plasminogen. No unique peptides were found. These studies suggest that apolipoprotein (a) could have arisen through duplication of specific regions from the human plasminogen gene. The size heterogeneity of apo (a) might then be explained by differences in the numbers of gene duplications.     

The primary structure of calf chymosin.

The complete amino acid sequence of calf chymosin (rennin) (EC 3.4.23.4) has been determined. The sequence consists of a single peptide chain of 323 amino acid residues. The primary structure of the precursor part of calf prochymosin was published previously (Pedersen, V.B., and Foltmann, B. (1975) Eur. J. Biochem. 55, 95-103), thus we are now able to account for the total 365 amino acid residues of calf prochymosin. Comparison of the sequence of calf prochymosin with that of pig pepsinogen A (EC 3.4.23.1) shows extensive homology. In the precursor part of the sequence, 15 residues are located at identical positions, as compared to 189 identical residues in the respective enzymes. Furthermore comparison to Penicillium janthinellum acid proteinase (penicillopepsin) (EC 3.4.23.7) shows that 76 residues are common to this enzyme and to the two gastric proteinases. These homologies in sequence further suggest that the folding of the peptide chain in chymosin is very similar to that of other acid proteinases.     

The complete primary structure of the alpha 2 chain of human type IV collagen and comparison with the alpha 1(IV) chain

The complete primary structure of the human type IV collagen alpha 2(IV) chain has been determined by nucleotide sequencing of cDNA clones. The overlapping cDNA clones cover 6,257 base pairs with a 5'-untranslated region of 283 base pairs, the 5,136-base pair open reading frame, and the 3'-untranslated region of 838 base pairs. The predicted amino acid sequence demonstrates that the complete translation product consists of 1,712 residues corresponding in molecular weight to 167,560. The translated polypeptide has a signal peptide of 36 amino acids, an amino-terminal noncollagenous part of 21 residues, a 1,428-residue collagenous domain with 23 interruptions, and a carboxyl-terminal noncollagenous (NC) domain of 227 residues. The calculated molecular mass of the mature human alpha 2(IV) chain is 163,774 Da.     

The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs.

Human liver cDNA coding for protein C has been synthesized, cloned and sequenced. The abundance of protein C message is approximately 0.02% of total mRNA. Three overlapping clones contain 1,798 nucleotides of contiguous sequence, which approximates the size of the protein's mRNA, based upon Northern hybridization. The cDNA sequence consists of 73 5'-noncoding bases, coding sequence for a 461 amino acid nascent polypeptide precursor, a TAA termination codon, 296 3'-noncoding bases, and a 38 base polyadenylation segment. The nascent protein consists of a 33 amino acid "signal", a 9 amino acid propeptide, a 155 amino acid "light" chain, a Lys-Arg connecting dipeptide, and a 262 amino acid "heavy" chain. Human protein C and Factor IX and X precursors possess about one third identical amino acids (59% in the gamma-carboxyglutamate domain), including two forty-six amino acid segments homologous to epidermal growth factor. Human protein C also has similar homology with prothrombin in the "leader", gamma-carboxyglutamate and serine protease domains, but lacks the two "kringle" domains found in prothrombin.     

Isolation and complete amino-acid sequence of the small polypeptide from light-harvesting pigment-protein complex I (B870) of Rhodopseudomonas capsulata

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.     

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, IV. The amino acid sequence of the human urinary trypsin inhibitor isolated by affinity chromatography

An acid-resistant trypsin inhibitor from human urine and serum is released in vivo by limited proteolysis from the high molecular acid-labile inter-alpha-trypsin inhibitor. The inhibitor shows an apparent molecular mass of 30 000 Da and is composed of two Kunitz-type domains. The domains are released in vitro by prolonged tryptic hydrolysis. The C-terminal domain is responsible for antitryptic activity. For the other domain no inhibitory activity towards proteinases, i.e. chymotrypsin, trypsin, pancreatic and leucocytic elastase has been demonstrated so far. The polypeptide chain comprising both domains consists of 122 residues and has a molecular mass of only 13 400 Da. In this work we have found that both, the N-terminal extension peptide with 21 residues and the "inactive" domain are linked O-glycosidically and N-glycosidically, respectively, with large carbohydrate moieties. The N-terminal amino acid sequence of the human urinary trypsin inhibitor was determined by solid-phase Edman degradation of a single peptide. The molecular mass calculated for the total polypeptide chain of 143 residues should be 15 340 Da; from the difference to the measured value (30 000 Da) it is concluded that the glycopeptide contains a considerable carbohydrate moiety.     

Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by B?hmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.     

Structure of the spectrin-actin binding site of erythrocyte protein 4.1

The complete primary structure of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations has been determined. The sequence of this domain, which contains 67 amino acids and has a molecular mass of 8045 daltons, has been obtained by NH2-terminal sequence analysis of an 8-kDa chymotryptic peptide, three endoproteinase lysine C-cleaved peptides and two peptides obtained by Staphylococcus aureus protease V8 cleavage. All peptides including the 8-kDa domain peptide were purified by reverse-phase high performance liquid chromatography. Antibodies against two different synthetic peptides of the 8-kDa domain are able to inhibit the association between protein 4.1, spectrin, and F-actin, corroborating that the 8-kDa domain is responsible for the formation of a ternary complex. A computer search of the 8-kDa sequence with the National Biomedical Research Foundation database did not detect any significant homologies to known sequences. Protein 4.1 is not related to any known proteins and may represent a new protein superfamily.     

The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin

The complete amino acid sequence of human insulin-like growth factor I (IGF-I), a polypeptide isolated from serum, has been determined. IGF-I is a single chain polypeptide of 70 amino acid residues cross-linked by three disulfide bridges. The calculated molecular weight is 7649. IGF-I displays obvious homology to proinsulin: positions 1 to 29 are homologous to insulin B chain and positions 42 to 62 to insulin A chain. A shortened "connecting" peptide with 12 residues (positions 30 to 41) compared to 30 to 35 in proinsulins shows no homology to proinsulin C peptide. An octapeptide sequence at the COOH-terminal end is also a feature not found in proinsulins. The number of differences in amino acid positions between IGF-I and insulins suggests that duplication of the gene of the common ancestor of proinsulin and IGF occurred before the time of appearance of the vertebrates. Of the 19 residues known to be invariant in all insulins so far sequenced, only glutamine A5 and asparagine A21 are replaced in IGF-I by glutamic acid and alanine, respectively. The fact that all half-cystine and glycine residues and most nonpolar core residues of the insulin monomer are conserved is compatible with a three-dimensional structure of IGF-I similar to that of insulin.     

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.     

The primary structure of staphylococcal protease.

The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protease B and the trypsin-like enzyme of Streptomyces griseus.     

Nucleotide sequence of cloned cDNA coding for preproricin

The primary structure of a precursor protein that contains the toxic (A) and galactose-binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)-rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24-amino-acid N-terminal signal sequence (molecular mass 2836 Da) preceding the A chain 267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12-amino-acid linking region (molecular mass 1385 Da).     

Studies on cytochrome c oxidase, IX. The primary structure of polypeptide VIa

The complete amino acid sequence of the cytoplasmic polypeptide VIa of cytochrome c oxidase from beef heart is described. The primary structure of this component of complex IV of the respiratory chain is elucidated by isolation and sequencing of overlapping glutamic acid, arginine, tryptophan and methionine fragments obtained by cleavage with Staphylococcus aureus protease, protease from submaxillaris glands of mice, 2-iodosylbenzoic acid and cyanogen bromide. The chain length of polypeptide VIa is 98 amino acids, the resulting molecular mass of 10670 Da. The hydrophilic protein does not contain a hydrophobic membrane penetrating sequence domain. Its function in the respiratory complex IV is unknown.     

Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.     

The mechanism of activation of rabbit plasminogen by urokinase.

The data presented in this paper show that when rabbit plasminogen is activated to plasmin by urokinase at least two peptide bonds are cleaved in the process. Urokinase first cleaves an internal peptide bond in plasminogen, leading to two-chain disulfide-linked plasmin molecule. The plasmin heavy chain of molecular weight 66,000 to 69,000 possesses an NH2-terminal amino acid sequence identical with the original plasminogen (molecular weight 88,000 to 92,000). The plasmin light chain of molecular weight 24,000 to 26,000 is known to be derived from the COOH-terminal portion of plasminogen. The plasmin generated during the activation of plasminogen is capable, by a feedback process, of cleaving a peptide of molecular weight 6,000 to 8,000 from the NH2 terminus of the heavy chain, producing a proteolytically modified heavy chain of molecular weight 58,000 to 62,000. Plasmin also can cleave this same peptide from the original plasminogen, yielding an altered plasminogen of molecular weight 82,000 to 86,000. This plasmin-altered plasminogen and the plasmin heavy chain derived from it by urokinase activation process NH2-terminal amino acid sequences which are identical with each other and with the plasminolytic product of the original plasmin heavy chain. These studies support a mechanism of activation of plasminogen by urokinase which involves loss of a peptide located on the NH2 terminus of plasminogen. However, these same results show that this NH2-terminal peptide need not be released from rabbit plasminogen prior to the cleavage of the internal peptide bond which leads to the two-chain plasmin molecule. Furthermore, these studies show that urokinase cannot remove this peptide from either the original rabbit plasminogen molecule or from the heavy chain of the initial plasmin formed.     

cDNA for the carboxyl-terminal region of a rat intestinal mucin-like peptide.

When subjected to thiol reduction, purified intestinal mucins have been shown to undergo a decrease in molecular mass and to liberate a 118-kDa glycopeptide (Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R., Bennick, A., Kawagishi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, 637-647). The latter has been called a putative "link" component because it is assumed to be important for disulfide bond-mediated mucin polymerization. Controversy exists as to whether the putative link is an integral mucin component or a separate mucin-associated glycopeptide. In the present study both NH2-terminal and internal amino acid sequences of the 118-kDa glycopeptide of rat intestinal mucin were used to generate opposing oligonucleotide primers for polymerase chain reaction. A specific 1.2-kilobase (kb) product was obtained, from which a 0.5-kb HindIII fragment was used as a probe to screen a lambda ZAP II cDNA library of rat intestine. A 2.6-kb cDNA (designated MLP 2677) was sequenced and revealed an open reading frame of 2.5 kb encoding 837 amino acids. The deduced amino acid sequence showed that the putative link peptide is equivalent to the carboxyl-terminal 689 amino acids of a larger peptide. Northern blots revealed a mRNA size of approximately 9 kb. Computer searches revealed no sequence homology with other proteins, but similarities were seen in the alignment of cysteine residues in the link and in several domains of human von Willebrand factor, as well as cysteine-rich areas of bovine and porcine submaxillary mucins and a frog skin mucin designated FIM-B.1. In keeping with earlier demonstrations of the presence of mannose in the 118-kDa glycopeptide, there were several (13) consensus sequences for attachment of N-linked oligosaccharides within the link domain. Further sequencing of MLP 2677 in a direction 5' to the codon specifying the NH2-terminal proline of the link has revealed a coding region for 148 amino acids, including a unique 75-amino acid domain rich in cysteine and proline, and a region containing 4.5-variable tandem repeats (each 11-12 amino acids) rich in serine, threonine, and proline. The presence of mucin-like tandem repeats suggests that the entire cysteine-rich link peptide represents the carboxyl-terminal region (75.5 kDa) of a mucin-like peptide (MLP). The latter is estimated to have a molecular mass of approximately 300 kDa.     

The isolation and characterization of the cyanogen bromide peptides from the B chain of human collagen.

Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.