Internal duplication and evolution of human ceruloplasmin.

With the completion of the primary structure of the 50,000- and 19,000-dalton fragments of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1], over half of the covalent structure of the single polypeptide chain of this protein is known. Visual and computer analysis of the sequence of the 564 amino acid residues in the two fragments gives clear evidence of statistically significant internal homology suggestive of evolutionary replication of two smaller units. Two homology regions, each composed of 224 residues, were defined by an intrasequence alignment that required only three gaps in each 224-residue segment. The two homology regions exhibited 43% identity in sequence, and 13% of the remaining positions had similar residues. The sequence of a 160-residue segment in ceruloplasmin exhibits significant homology to the active (copper-binding) sites of blue electron-transfer proteins such as azurins and plastocyanins and multicopper oxidases such as cytochrome oxidase and superoxide dismutase. It is proposed that a primitive ceruloplasmin gene was formed by the fusion of two genes coding, respectively, for protein abut 160 and 190 amino acid residues in length and that this precursor gene coding for about 350 amino acids was later triplicated to form the gene for the present-day ceruloplasmin molecule of about 1050 amino acids.     

Internal triplication in the structure of human ceruloplasmin.

Amino acid sequence analysis of the 67,000-dalton (67-kDal) fragment that is the amino-terminal half of human ceruloplasmin has revealed internal triplication in the primary structure of the entire molecule. This is illustrated by comparison of 620 residues representing homologous domains of the 67-kDal fragment and of the 50-kDal and 19-kDal fragments that together comprise the carboxyl-terminal half of the molecule. The polypeptide chain is divided into three covalently linked homologous segments, each of about 340 residues. All three homology units have about 30% identity in sequence, and each pair exhibits at least 40% identity. The statistical significance of the 3-fold internal duplication was established by computerized analysis of the sequence. These results and studies of the sites of limited proteolytic cleavage support a model for the ceruloplasmin molecule consisting of an alternating structure of six domains of two different kinds (or possibly nine domains of three kinds). The 3-fold internal homology suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes.     

Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum.

The complete primary structure of the 3.1S leucine-rich alpha 2-glycoprotein (LRG) present in human plasma has been determined. This protein (Mr approximately 45,000) consists of a single polypeptide chain with one galactosamine and four glucosamine oligosaccharides attached. The polypeptide has two intrachain disulfide bonds and contains 312 amino acid residues of which 66 are leucine. The amino acid sequence can be exactly divided into 13 segments of 24 residues each, eight of which exhibit a periodic pattern in the occurrence of leucine, proline, and asparagine. The consensus sequence for the repeating tetracosapeptide unit is Pro-Xaa-Xaa-Leu-Leu-Xaa-Xaa-Xaa-X aa-Xaa-Leu-Xaa-Xaa-Leu-Xaa-Leu-Xaa-Xaa-Asn-Xaa-Leu-Xaa-Xaa-Leu. This periodicity suggests that the unique structure of LRG arose from a series of unequal crossovers of a precursor oligonucleotide sequence that encoded a building block rich in leucine. Overall, the amino acid sequence of LRG is not significantly homologous to the continuous sequence of any protein in the current data base. However, the consensus tetracosapeptide sequence shows strong homology to segments of many mitochondrial proteins, viral envelope proteins, and oncogene proteins that have a high leucine content and transmembrane domains. Tandem repetition of similar segments also occurs in apolipoproteins that have amphipathic helical potential. Prediction of the secondary structure by the Chou-Fasman rules and calculation of the hydrophilic/hydrophobic profile by several methods confirm the tandem repetition of largely hydrophobic structural units; these begin with a beta-turn that leads into an organized structure with alpha-helical or beta-sheet potential. These structural characteristics and the homology to mitochondrial proteins and apolipoproteins suggest that LRG is a membrane-derived or membrane-associated protein containing a series of domains capable of bipolar surface orientation.     

Complete amino acid sequence of human hemopexin, the heme-binding protein of serum.

We have determined the complete primary structure of human hemopexin, a plasma beta-glycoprotein that specifically binds one heme with high affinity and transports it to hepatocytes for salvage of the iron. Human hemopexin (Mr approximately equal to 63,000) consists of a single polypeptide chain containing 439 amino acid residues with six intrachain disulfide bridges. The amino-terminal threonine residue is blocked by an O-linked galactosamine oligosaccharide, and the protein has five glucosamine oligosaccharides N-linked to the acceptor sequence Asn-X-Ser/Thr. The 18 tryptophan residues are arranged in four clusters, and 12 of the tryptophans are conserved in homologous positions. Computer-assisted analysis of the internal homology in amino acid sequence indicates that hemopexin consists of two similar halves, thus suggesting duplication of an ancestral gene. Limited tryptic digestion cleaves apohemopexin after arginine-216 into two half-molecules, whereas heme-saturated hemopexin is cleaved after lysine-101. The half-molecules are connected by a histidine-rich hinge-like region that contains two glucosamine oligosaccharides. A structural model for human hemopexin is proposed that is based on these properties and on computer-assisted predictions of the secondary structure and the hydrophilic/hydrophobic character. In this model alpha-helices and beta-turns predominate, and the two halves are connected by an exposed connecting region in apohemopexin that becomes inaccessible to trypsin in hemesaturated hemopexin. Many segments of hemopexin are similar to sequences of other heme proteins, but no overall structural relationship of hemopexin to any other heme protein was identified.     

Complete amino acid sequence of a 50,000-dalton fragment of human ceruloplasmin.

The complete amino acid sequence has been determined for a 50,000-dalton fragment that is an internal segment of the single polypeptide chain of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F4) contains 405 amino acid residues, has one glucosamine-containing carbohydrate unit, and, together with the 19,000-dalton fragment that follows it, accounts for the carboxyl-terminal half of the molecule. Fragment Cp F4 has a very nonuniform distribution of certain amino acid residues, which show a high potential to be adjacent to or one residue separated from a similar amino acid. This is most pronounced for acid and amide residues (65% clustered), aromatic residues (56% clustered), and basic residues (41% clustered). In addition, there is a long-range clustering of proline residues at the amino- and carboxyl-terminal 60 residues (50% clustered). Also, there are a number of short repeated segments of sequence. Calculations based on parameters predictive of secondary structure folding patterns indicate that the 50,000-dalton fragment has a low content of alpha-helix and is predominantly beta-sheet, beta-turn, and random in structure. Limited enzymatic cleavage of human ceruloplasmin to yield 67,000-, 50,000-, and 19,000-dalton fragments occurs at specific exposed sites of random structure in between domain-like regions.     

Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.

The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.     

Amino acid sequence of human plasma alpha 1B-glycoprotein: homology to the immunoglobulin supergene family.

The complete amino acid sequence has been determined for alpha 1B-glycoprotein (alpha 1B), a protein of unknown function present in human plasma. This protein (Mr approximately equal to 63,000) consists of a single polypeptide chain N-linked to four glucosamine oligosaccharides. The polypeptide has five intrachain disulfide bonds and contains 474 amino acid residues. Analysis of the amino acid sequence by several computer programs shows that alpha 1B exhibits internal duplication and consists of five repeating structural domains, each containing about 95 amino acids and one disulfide bond. alpha 1B has a unique amino acid sequence. However, several domains of alpha 1B, especially the third, show statistically significant homology to variable regions of certain immunoglobulin light and heavy chains. alpha 1B also exhibits sequence similarity to other members of the immunoglobulin supergene family such as the receptor for transepithelial transport of IgA and IgM and the secretory component of human IgA. Because of its internal duplication and its sequence homology to immunoglobulin-like proteins, alpha 1B appears to have evolved from an ancestral gene similar to that of the immunoglobulin supergene family.     

Single-chain structure of human ceruloplasmin: the complete amino acid sequence of the whole molecule.

We have determined the amino acid sequence of the amino-terminal 67,000-dalton (67-kDa) fragment of human ceruloplasmin and have established overlapping sequences between the 67-kDa and 50-kDa fragments and between the 50-kDa and 19-kDa fragments. The 67-kDa fragment contains 480 amino acid residues and three glucosamine oligosaccharides. These results together with our previous sequence data for the 50-kDa and 19-kDa fragments complete the amino acid sequence of human ceruloplasmin. The polypeptide chain has a total of 1,046 amino acid residues (Mr 120,085) and has attachment sites for four glucosamine oligosaccharides; together these account for the total molecular mass of human ceruloplasmin (132 kDa). The sequence analysis of the peptides overlapping the fragments showed that one additional amino acid, arginine, is present between the 67-kDa and 50-kDa fragments, and another, lysine, is between the 50-kDa and 19-kDa fragments. Only two apparent sites of amino acid interchange have been identified in the polypeptide chain. Both involve a single-point interchange of glycine and lysine that would result in a difference in charge. The results of the complete sequence analysis verified that human ceruloplasmin is composed of a single polypeptide chain and that the subunit-like fragments are produced by proteolytic cleavage during purification (and possibly also in vivo).     

Amino acid sequence of porcine spleen cathepsin D.

The amino acid sequence of porcine spleen cathepsin D heavy chain has been determined and, hence, the complete structure of this enzyme is now known. The sequence of heavy chain was constructed by aligning the structures of peptides generated by cyanogen bromide, trypsin, and endo-proteinase Lys C cleavages. The structure of the light chain has been published previously. The cathepsin D molecule contains 339 amino acid residues in two polypeptide chains: a 97-residue light chain and a 242-residue heavy chain, with a combined Mr of 36,779 (without carbohydrate). There are two carbohydrate units linked to asparagine residues 70 and 192. The disulfide bond arrangement in cathepsin D is probably similar to that of pepsin, because the positions of six half-cystine residues are conserved. The active site aspartyl residues, corresponding to aspartic acid-32 and -215 of pepsin, are located at residues 33 and 224 in the cathepsin D molecule. The amino acid sequence around these aspartyl residues is strongly conserved. Cathepsin D shows a strong homology with other acid proteases. When the sequence of cathepsin D, renin, and pepsin are aligned, 32.7% of the residues are identical. The homology is observed throughout the length of the molecules, indicating that three-dimensional structures of all three molecules are similar.     

Primary structure of major outer membrane protein I of Escherichia coli B/r.

The amino acid sequence of the pore-forming outer membrane protein I (porin) from Escherichia coli B/r has been determined. The polypeptide contains 340 amino acid residues resulting in a molecular weight of 37,205. The transmembrane polypeptide has no stretches of nonpolar residues, uninterrupted by charged side chains, longer than 11 amino acid residues. Regarding polarity, the chain can be subdivided into three regions: a distinctly hydrophilic region between residues 1 and 82 (51.2% polarity), a fairly nonpolar region between residues 83 and 194 (33.9% polarity), and a more hydrophilic region up to the COOH terminus (48% polarity). These results are interpreted as evidence against a simple transmembrane structure in which the membrane is spanned by a single contiguous sequence of hydrophobic amino acids, as has been proposed, for example, for glycophorin.     

The complete amino acid sequence of human serum transferrin.

The complete amino acid sequence of human serum transferrin has been determined by aligning the structures of the 10 CNBr fragments. The order of these fragments in the polypeptide chain is deduced from the structures of peptides overlapping methionine residues and other evidence. Human transferrin contains 678 amino acid residues and--including the two asparagine-linked glycans--has an overall molecular weight of 79,550. The polypeptide chain contains two homologous domains consisting of residues 1-336 and 337-678, in which 40% of the residues are identical when aligned by inserting gaps at appropriate positions. Disulfide bond arrangements indicate that there are seven residues between the last half-cystine in the first domain and the first half-cystine in the second domain and therefore, a maximum of seven residues in the region of polypeptide between the two domains. Transferrin--which contains two Fe-binding sites--has clearly evolved by the contiguous duplication of the structural gene for an ancestral protein that had a single Fe-binding site and contained approximately 340 amino acid residues. The two domains show some interesting differences including the presence of both N-linked glycan moieties in the COOH-terminal domain at positions 413 and 610 and the presence of more disulfide bonds in the COOH-terminal domain (11 compared to 8). The locations of residues that may function in Fe-binding are discussed.     

Complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein and its homology to histocompatibility antigens

In the present study the complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein was determined. This protein whose biological function is unknown consists of a single polypeptide chain of 276 amino acid residues including 8 tryptophan residues and has a pyroglutamyl residue at the amino terminus. The location of the two disulfide bonds in the polypeptide chain was also established. The three glycans, whose structure was elucidated with the aid of 500 MHz 1H NMR spectroscopy, were sialylated N-biantennas. The molecular weight calculated from the polypeptide and carbohydrate structure is 38,478, which is close to the reported value of approximately equal to 41,000 based on physicochemical measurements. The predicted secondary structure appeared to be comprised of 23% alpha-helix, 27% beta-sheet, and 22% beta-turns. The three N-glycans were found to be located in beta-turn regions. An unexpected finding was made by computer analysis of the sequence data; this revealed that Zn-alpha 2-glycoprotein is closely related to antigens of the major histocompatibility complex in amino acid sequence and in domain structure. There was an unusually high degree of sequence homology with the alpha chains of class I histocompatibility antigens. Moreover, this plasma protein was shown to be a member of the immunoglobulin gene superfamily. Zn-alpha 2-glycoprotein appears to be a truncated secretory major histocompatibility complex-related molecule, and it may have a role in the expression of the immune response.     

Nucleotide sequence of the structural gene for colicin E1 and predicted structure of the protein.

We have determined the nucleotide sequence of the structural gene for colicin E1, which consists of 1,566 base pairs. The amino acid sequence (522 residues) of the protein was derived from the DNA sequence, and the molecular weight was calculated to be 57,279. From the analysis of the predicted secondary structure, there appear to be three consecutive long alpha-helices in the NH2-terminal half of the polypeptide, spanning 40, 100, and 35 amino acid residues. In addition, there is a polypeptide region near the COOH terminus that shows homology to the NH2-terminal signal portions of outer membrane lipoprotein in Escherichia coli and beta-lactamase in Bacillus licheniformis. Most of the homologous amino acids are located in the region where either alpha-helix or beta-sheet would be expected to occur, as determined from the amino acid sequence. These characteristics of the predicted protein structure might correspond to properties of colicin E1 as an ionophore in its antimicrobial action and also as an exported protein during its induced synthesis.     

THE COVALENT STRUCTURE OF AN ENTIRE γG IMMUNOGLOBULIN MOLECULE

The complete amino acid sequence of a human gammaG1 immunoglobulin (Eu) has been determined and the arrangement of all of the disulfide bonds has been established. Comparison of the sequence with that of another myeloma protein (He) suggests that the variable regions of heavy and light chains are homologous and similar in length. The constant portion of the heavy chain contains three homology regions each of which is similar in size and homologous to the constant region of the light chain. Each variable region and each constant homology region contains one intrachain disulfide bond. The half-cystines participating in the interchain bonds are all clustered within a stretch of ten residues at the middle of the heavy chains.These data support the hypothesis that immunoglobulins evolved by gene duplication after early divergence of V genes, which specified antigen-binding functions, and C genes, which specified other functions of antibody molecules. Each polypeptide chain may therefore be specified by two genes, V and C, which are fused to form a single gene (translocation hypothesis). The internal homologies and symmetry of the molecule suggest that homology regions may have similar three-dimensional structures each consisting of a compact domain which contributes to at least one active site (domain hypothesis). Both hypotheses are in accord with the linear regional differential of function in antibody molecules.     

Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides.

The tendency of a polypeptide chain to form alpha-helical or beta-strand secondary structure depends upon local and nonlocal effects. Local effects reflect the intrinsic propensities of the amino acid residues for particular secondary structures, while nonlocal effects reflect the positioning of the individual residues in the context of the entire amino acid sequence. In particular, the periodicity of polar and nonpolar residues specifies whether a given sequence is consistent with amphiphilic alpha-helices or beta-strands. The importance of intrinsic propensities was compared to that of polar/nonpolar periodicity by a direct competition. Synthetic peptides were designed using residues with intrinsic propensities that favored one or the other type of secondary structure. The polar/nonpolar periodicities of the peptides were designed either to be consistent with the secondary structure favored by the intrinsic propensities of the component residues or in other cases to oppose these intrinsic propensities. Characterization of the synthetic peptides demonstrated that in all cases the observed secondary structure correlates with the periodicity of the peptide sequence--even when this secondary structure differs from that predicted from the intrinsic propensities of the component amino acids. The observed secondary structures are concentration dependent, indicating that oligomerization of the amphiphilic peptides is responsible for the observed secondary structures. Thus, for self-assembling oligomeric peptides, the polar/nonpolar periodicity can overwhelm the intrinsic propensities of the amino acid residues and serves as the major determinant of peptide secondary structure.     

Isolation and sequence of a human cytochrome P-450 cDNA clone.

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.     

Primary structure of rat thymus prothymosin alpha.

The primary structure of prothymosin alpha from rat thymus, containing 113 amino acid residues, is reported as follows: (formula; see text) The sequence of the first 28 amino acids at the NH2 terminus is identical to that of calf thymosin alpha 1. The dicarboxylic amino acids, which account for nearly half of the total residues in prothymosin alpha, are largely clustered in the central portion of the polypeptide chain. The polypeptide contains no aromatic or sulfur-containing amino acids. A computer analysis of the three-dimensional structure based on the primary sequence suggests that the molecule is composed of at least five alpha-helical regions interrupted by one short extended chain and three short random coils.     

Epitope mapping of human factor VIII inhibitor antibodies by site-directed mutagenesis of a factor VIII polypeptide.

Previous epitope mapping studies of human factor VIII (FVIII) inhibitor antibodies with heavy chain specificity localized epitopes to the amino-terminal half of the FVIII A2 domain. In this report we have used unidirectional deletion analysis and site-directed mutagenesis to identify a minimum length polypeptide and amino acid residues that contribute to the FVIII conformation recognized by these antibodies. Bacterial expression plasmids were exploited to demonstrate that a FVIII polypeptide of approximately 150 residues is required to generate a common heavy chain epitope(s). Another series of plasmids were constructed that synthesize: a FVIII polypeptide containing an internal deletion; four polypeptides with single residue substitutions; two polypeptides with triple residue changes; and a quadruple amino acid replacement within one polypeptide. The relative reactivities of the wild-type and mutant FVIII polypeptides were tested by immunoblotting, inhibitor neutralization assays and ELISA with a variety of human FVIII inhibitor auto- and alloantibodies. These techniques illustrate that the internal deletion mutant and one of the relatively conservative amino acid substitution triple mutants, mutant 389, resulted in significantly decreased immunoreactivity. The data identify FVIII Glu389,390,391 as three critical components of an epitope for human FVIII inhibitor antibodies and identify a major inhibitory epitope involved in the immune response to FVIII.     

Homologous inhibitors from potato tubers of serine endopeptidases and metallocarboxypeptidases.

A potent polypeptide inhibitor of chymotrypsin has been purified from Russett Burbank potatoes. The inhibitor has no effect on bovine carboxypeptidases A or B but exhibits homology with a carboxypeptidase inhibitor that is also present in potato tubers. The chymotrypsin inhibitor has a molecular weight of approximately 5400 as estimated by gel filtration, amino acid analysis, and titration with chymotrypsin. The polypeptide chain consists of 49 amino acid residues, of which six are half-cystine, forming three disulfide bonds. Its size is similar to that of the carboxypeptidase inhibitor, which contains 39 amino acid residues and also has three disulfide bridges. In immunological double diffusion assays, the chymotrypsin inhibitor and the carboxypeptidase inhibitor do not crossreact; however, automatic Edman degradation of reduced and alkylated derivatives of the chymotrypsin inhibitor, yielding a partial sequence of 18 amino acid residues at the NH2-terminus, reveals a similarity in sequence to that of the carboxypeptidase inhibitor. Thus, inhibitors directed toward two distinct classes of proteases, the serine endopeptidases and the metallocarboxypeptidases, appear to have evolved from a common ancestor.     

Primary structure of blood coagulation factor XIIIa (fibrinoligase, transglutaminase) from human placenta.

We have determined the primary structure of human placental factor XIIIa, an enzyme [fibrinoligase, transglutaminase, fibrin-stabilizing factor, EC 2.3.2.13 (protein-glutamine:amine gamma-glutamyltransferase)] that forms intermolecular isopeptide bonds between fibrin molecules as the last step in blood coagulation. Placental factor XIIIa is an unglycosylated polypeptide chain of 730 amino acid residues (Mr = 83,005) that appears to be identical to the a subunit of the plasma zymogen factor XIII. Ca2+-dependent activation of factor XIIIa by thrombin removes a blocked amino-terminal peptide and unmasks a reactive thiol group at Cys-314. A second specific cleavage after Lys-513 by thrombin inactivates factor XIIIa and produces an amino-terminal 56-kDa fragment and a 24-kDa fragment. The amino acid sequence of factor XIIIa is unique and does not exhibit internal homology, but its active center is similar to that of the thiol proteases. The probable Ca2+-binding site of factor XIIIa has been identified by homology to the high-affinity sites in calmodulins. Knowledge of the primary structure of factor XIIIa will aid elucidation of the mechanism of its enzymatic action and that of the many tissue transglutaminases of which it is the prototype. This will also facilitate production of factor XIIIa by recombinant DNA technology for use in treatment of congenital factor XIII deficiencies and in the postoperative healing of wounds.