Amyloid-related serum protein SAA from three animal species: comparison with human SAA.

The amyloid-relates serum protein SAA has been isolated by gel filtration in 10% formic acid from three animal species: mink, mouse, rabbit. Sera used in the isolation procedure were obtained from animals in which high concentrations of SAA had been induced by treatment with LPS. The isolated SAA proteins had a subunit size similar to that of human SAA, with m.w. values ranging from 10,000 to 11,700 (estimated by gel filtration in 6 M guanidine-HC1) or 12,400 to 15,000 (estimated by SDS-PAGE). The m.w. studies and amino acid sequence data indicated that SAA and the amyloid fibril protein AA in the mouse, and probably also the mink, are related in the same way as in man, the two proteins having common NH2-terminal amino acid sequences and SAA being extended by 20 to 40 residues at the COOH-terminal end of the molecule.     

Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences

The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.     

Nuclear genes for cytochrome c oxidase subunits of Neurospora crassa. Isolation and characterization of cDNA clones for subunits IV, V, VI, and possibly VII

We obtained cDNA clones for cytochrome oxidase subunits IV, V, VI, and possibly VII by constructing a lambda gt11 library of Neurospora crassa cDNA and probing it with antiserum directed against Neurospora cytochrome oxidase holoenzyme. Positive clones were further characterized with antisera directed against individual cytochrome oxidase subunits and subsequently by DNA sequencing. The clones for subunits IV and V encode proteins with regions matching the known N-terminal amino acid sequences of purified Neurospora cytochrome oxidase subunits IV and V, respectively. The sequences of these clones provide the first evidence that cytochrome oxidase subunits IV and V are made as precursors with N-terminal extensions in Neurospora. The N-terminal extensions encoded by these clones share homology, and are rich in arginine, as are signal sequences of other mitochondrially destined proteins. The subunit VI clone codes for the carboxyl terminus of a protein homologous to the carboxy termini of yeast cytochrome oxidase subunit VI and bovine cytochrome oxidase subunit Va. The subunit VII clone contains an open reading frame for a 47-residue protein, the expected size for subunit VII. However, the protein coded by this clone has an unusual amino acid composition. Whether this clone represents an authentic cytochrome oxidase subunit is not established.     

Basic proline-rich proteins from human parotid saliva: relationships of the covalent structures of ten proteins from a single individual.

Eleven basic proline-rich proteins were purified from the parotid saliva of a single individual. The complete amino acid sequences of six of these were determined by conventional protein sequence methodology, bringing to nine the number of known primary structures of nonglycosylated basic proline-rich proteins from the same individual. The partial sequence of one additional protein is also reported. All of the basic proline-rich proteins studied contain segments with identical or very similar sequences, but with two possible exceptions, none of the proteins is derived from another secreted proline-rich protein. The amino acid sequences of nine nonglycosylated basic proline-rich proteins were compared with primary structures deduced from published nucleotide sequences of DNA coding for human parotid proline-rich proteins. The sequences align well, in general, but differences also exist pointing to the complexity of the genetics of these proteins. Seven secretory basic proline-rich proteins appear to be formed from three larger precursors by selective posttranslational proteolyses of arginyl bonds. One of the basic proline-rich proteins appears to derive from human acidic proline-rich proteins. The remaining two proteins studied do not conform to any DNA structure as yet reported. Two of the basic proline-rich proteins studied are phosphoproteins and exhibit abilities to inhibit hydroxyapatite formation in vitro.     

cDNA sequences of two apolipoproteins from lamprey

The messages for two small but abundant apolipoproteins found in lamprey blood plasma were cloned with the aid of oligonucleotide probes based on amino-terminal sequences. In both cases, numerous clones were identified in a lamprey liver cDNA library, consistent with the great abundance of these proteins in lamprey blood. One of the cDNAs (LAL1) has a coding region of 105 amino acids that corresponds to a 21-residue signal peptide, a putative 8-residue propeptide, and the 76-residue mature protein found in blood. The other cDNA (LAL2) codes for a total of 191 residues, the first 23 of which constitute a signal peptide. The two proteins, which occur in the "high-density lipoprotein fraction" of ultracentrifuged plasma, have amino acid compositions similar to those of apolipoproteins found in mammalian blood; computer analysis indicates that the sequences are largely helix-permissive. When the sequences were searched against an amino acid sequence data base, rat apolipoprotein IV was the best matching candidate in both cases. Although a reasonable alignment can be made with that sequence and LAL1, definitive assignment of the two lamprey proteins to typical mammalian classes cannot be made at this point.     

Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea

Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor.     

The complete amino acid sequence of a biologically active 197-residue fragment of M protein isolated from type 5 group A streptococci

The complete amino acid sequence of a peptic fragment (Pep M5) of the group A streptococcal type 5 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment, comprising nearly half of the native M molecule, is biologically active in that it has the ability to interact with opsonic antibodies as well as to evoke such an antibody response in rabbits. The sequence of Pep M5 was determined by automated Edman degradations of the uncleaved molecule and its enzymatically derived peptides. The primary peptides for Edman degradation were the arginine peptides obtained by tryptic digestion. The tryptic cleavage of Pep M5 was limited to the arginyl peptide bonds by derivatizing the epsilon-amino groups of lysine residues by reductive dihydroxypropylation. The overlapping peptides were generated by digestion of the unmodified Pep M5 with chymotrypsin, V8 protease, and subtilisin. The sequence thus established for the Pep M5 molecule consists of a total of 197 residues (Mr = 22,705). The Pep M5 protein contains some identical, or nearly so, repeating sequences: four 7-residue segments and two 10-residue segments. However, extensive sequence repeats of the kind previously reported within the partial sequence of another M protein serotype, namely Pep M24, were absent. The Pep M5 sequence is distinct from, but exhibits some homology with, the partial sequences of two other M protein serotypes, namely, Pep M6 and Pep M24. Furthermore, the 7-residue periodicity of the nonpolar and charged residues, an alpha-helical coiled-coil structural characteristic that was previously observed within the partial sequences of M proteins, was found to extend over a significant part of the Pep M5 sequence. The implication of these results to the function and immunological diversity in M proteins is discussed.     

Yeast ribosomal proteins. VIII. Isolation of two proteins and sequence characterization of twenty-four proteins from cytoplasmic ribosomes

Summary Two proteins, YL41 and YL43, were isolated from 80S ribosomes of Saccharomyces cerevisiae by filtration through a Sephacryl S-200 column and by chromatography on a column of carboxymethylcellulose. Their amino acid compositions are presented. Twenty-four proteins including these two proteins were subjected to sequence analyses by automated Edman degradation. Amino-terminal amino acid sequences were determined for 17 proteins, YS3, YS9, YS23, YS24, YS29, YL6, YL8, YL11, YL15, YL17, YL23, YL28, YL33, YL37, YL39, YL41, and YL43. YL41, which has a 72.7% lysine and arginine content, was found to be particular to eukaryotic ribosomes. The aminotermini of another seven proteins, YS2, YS5, YS8, YS12, YS13, YS20, and YS27, were suggested to be blocked.Comparison of the amino-terminal sequences with all other ribosomal protein sequences so far available indicates that YS9 shows sequence homology to rat liver ribosomal protein S8 (Wittmann-Liebold et al. 1979).     

Polymorphism of human complement component C4

An assessment has been made of the polymorphism of human complement component C4 by comparing derived amino acid sequences of cDNA and genomic DNA with limited amino acid sequences. In all, one complete and six partial sequences have been obtained from material from three individuals and include two C4A and two C4B alleles. Differences were found between the 4 alleles from 2 loci in only 15 of the 1722 amino acid residues, and 12 lie within one section of 230 residues, which in 1 allele also contains a 3-residue deletion. In three variable positions, an allelic difference in one C4 type was common to the other types. Three nucleotide differences were found in four introns. In spite of marked differences in their chemical reactivity, the many allelic forms appear to differ in less than 1% of their amino acid residue positions. This unusual pattern of polymorphism may be due to recent duplication of the C4 gene, or may have arisen by selection as a result of the biological role of C4, which interacts in the complement sequence with nine other proteins necessitating conservation of much of the surface structure.     

Sequences of two expressed sequence tags (EST) from rice encoding different cap-binding proteins

Wheat has been shown to have two forms of the cap-binding protein that participate in the initiation of translation. To identify cap-binding proteins from other higher plant species, the expressed sequence tag (EST) database was searched. Several rice ESTs were identified with similarity to both forms of the wheat cap-binding proteins. Two of the rice ESTs were obtained and the cDNA sequences completed. The deduced amino acid sequences of the rice cap-binding proteins are compared to the wheat cap-binding proteins and cap-binding proteins from Saccharomyces cerevisiae, Drosophila melanogaster, Xenopus laevis and human.     

Tentative identification of a resilin gene in Drosophila melanogaster

A search of the Drosophila genome for gene products with similarities to the amino acid sequences of three tryptic peptides from locust (Schistocerca gregaria) resilin gave two positive results: gene products CG15920 and CG9036. In both conceptual translation products a 62-residue region is present, which is identical to the resilin peptides in 29 positions. Gene product CG15920 has an amino acid composition closely resembling that of resilins from various insect species, and it has an N-terminal signal peptide sequence indicating that it is an extracellular protein. The 62-residue region shows similarity to the RR-2 sequence, which is common for a number of matrix proteins from insect solid cuticle. The N- and C-terminal regions flanking the 62-residue in CG15920 are dominated by 18 repeats of a 15-residue sequence and 11 repeats of a 13-residue sequence, respectively. The structures of the repeats predict that the peptide chain will fold in an irregular, extended beta-spiral, resembling the structures suggested for mammalian elastin and spider flagelliform silk, two materials which, like resilin, possess long-range elasticity. Accordingly, we suggest that gene product CG15920 is a Drosophila resilin precursor.     

Purification and characterization of the 22-kilodalton potato tuber proteins

Three abundant proteins of approximate molecular masses of 22, 23, and 24 kilodaltons were purified from potato (Solanum tuberosum L.) tubers by DEAE cellulose and CM-52 cellulose ion exchange column chromatography, electroelution, and high-pressure liquid chromatography (HPLC). Antibodies specific to the gel-purified 22-kilodalton protein were prepared. Immunoblot analysis showed that the 22-, 23-, and 24-kilodalton proteins are immunologically related and that these proteins are present in tubers and as higher molecular mass forms in leaves, but not in stems, roots, and stolons. The ratios of amino acid composition were compared among the three purified proteins, and the aminoterminal amino acid sequences were determined for these three proteins. All three proteins have identical amino-terminal sequences that match the deduced amino acid sequence of an abundant tuber protein cDNA.     

Cloning and nucleotide sequencing of genes for a second type of small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus"

The nucleotide sequences of the single genes coding for the B-type small, acid-soluble spore proteins (SASP) of Bacillus cereus, B. stearothermophilus, and "Thermoactinomyces thalpophilus" were determined, and the amino acid sequences of all B-type SASP were compared. While this type of SASP showed significant sequence conservation around the two spore protease cleavage sites, alignment of these sequences required the introduction of gaps, and even then only 19 of the residues were conserved exactly in all five proteins. However, all five B-type SASP did contain a large (27 to 35-residue), rather well-conserved amino acid sequence repeat, and four of the five proteins had well-conserved regions of 14 to 17 amino acids which appeared three times.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.     

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)     

The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.     

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.     

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.     

Isolation of a Drosophila gene coding for a protein containing a novel phosphatidylserine-binding motif

To elucidate the molecular basis of the binding of proteins to the membrane phospholipid phosphatidylserine (PS), we characterized PS-binding peptides isolated from a phage display library. Amino acid sequences deduced from the nucleotide sequences of over 60 phage clones isolated revealed that there was no common primary structure among these peptides, but all peptides were rich in basic amino acid residues. In particular, 15 clones encoded peptides that contained contiguous arginine residues. Characterization of two such peptides in more detail showed that they bound to PS, and to a much lower extent to other phospholipids, including phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine. Unlike other Ca2+-dependent PS-binding proteins, these peptides did not require Ca2+ for binding to PS, and the addition of Ca2+ did not alter the phospholipid specificity. Substitution of one of the two RR sequences in one peptide by alanine had no effect, but that of both sequences completely abolished the activity. Furthermore, we identified a Drosophila gene coding for a presumed nuclear protein that shares an amino acid sequence, including a RR residue, with one of the two PS-binding peptides. This protein bound to PS partly depending on the presence of the RR residue. These results allowed us to conclude that an amino acid sequence including contiguous arginine residues is a novel motif that defines Ca2+-independent PS-binding activity.