Antigenic variation and the surface glycoproteins of Trypanosoma congolense

Two Trypanosoma congolense variant-specific glycoproteins, which are expressed sequentially during a relapsing infection, have been purified. The proteins, termed VSG-1 and VSG-2, both have a molecular weight of 53,000 as determined by SDS polyacrylamide electrophoresis. When either antigen is electrophoresed through a pH gradient on an isoelectric focusing (IEF) gel, it gives a characteristic spectrotype of three bands. The IEF components of each VSG are antigenically similar to each other but not identical. The components of VSG-1 are immunologically distinct from the components of VSG-2, as shown by lack of cross-reactivity. The three spectrotypes may reflect microheterogeneity in amino acid sequence among the components. Both VSG-1 and VSG-2 are selectively cleaved by trypsin near their carboxy-terminal ends, indicating the existence of a possible common VSG region. Significant homology in the aminoterminal amino acid sequences of VSG-1 and VSG-2 suggests that sequentially reduplicated genes are sequentially expressed by trypanosomes during relapsing infections.     

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: III. Immunochemical characterization

A radioimmunoassay (RIA) for the variant-specific glycoproteins (VSG-1 and VSG-2) of two sequentially appearing variants of Trypanosoma congolense has been devised. When the isoelectrically focused VSG-1 components (VSG-1a, VSG-1b, and VSG-1c) are used as inhibitors of the VSG-1-anti-VSG-1 interaction, the RIA inhibition curves resemble each other, although minor differences in the high-affinity region of the curves can be detected. The heterologous antigen (VSG-2) does not inhibit the VSG-1-anti-VSG-1 interaction except at very high concentrations, indicating there is little cross-reactivity between highly purified VSG-1 and VSG-2. Nevertheless, heterologous antiserum, directed against VSG-2, will inhibit the VSG-1 -anti-VSG-1 interaction, and this property is shared to a significant degree by rabbit antiserum directed against an unrelated antigen. We have interpreted these findings as suggesting that: (1) there may be a constant region common to both VSG proteins, and (2) the constant region of the immunoglobulin molecule may also bind VSG proteins. Preliminary experiments show that the VSG-1 molecule augments binding of the Clq component of complement to the Fc region of immunoglobulin G.     

The occurrence of two types of collagen proalpha-chain in the abalone Haliotis discus muscle.

Acid-soluble collagens were prepared from connective tissues in the abalone Haliotis discus foot and adductor muscles with limited proteolysis using pepsin. Collagen preparation solubilized with 1% pepsin contained two types of alpha-chains which were different in their N-terminal amino acid sequences. Accordingly, two types of full-length cDNAs coding for collagen proalpha-chains were isolated from the foot muscle of the same animal and these proteins were named Hdcols (Haliotis discus collagens) 1alpha and 2alpha. The two N-terminal amino acid sequences of the abalone pepsin-solubilized collagen preparation corresponded to either of the two sequences deduced from the cDNA clones. In addition, several tryptic peptides prepared from the pepsin-solubilized collagen and fractionated by HPLC showed N-terminal amino acid sequences identical to those deduced from the two cDNA clones. Hdcols 1alpha and 2alpha consisted of 1378 and 1439 amino acids, respectively, showing the primary structure typical to those of fibril-forming collagens. The N-terminal propeptides of the two collagen proalpha-chains contained cysteine-rich globular domains. It is of note that Hdcol 1alpha completely lacked a short Gly-X-Y triplet repeat sequence in its propeptide. An unusual structure such as this has never before been reported for any fibril-forming collagen. The main triple-helical domains for both chains consisted of 1014 amino acids, where a supposed glycine residue in the triplet at the 598th position from the N-terminus was replaced by alanine in Hdcol 1alpha and by serine in Hdcol 2alpha. Both proalpha-chains of abalone collagens contained six cysteine residues in the carboxyl-terminal propeptide, lacking two cysteine residues usually found in vertebrate collagens. Northern blot analysis demonstrated that the mRNA levels of Hdcols 1alpha and 2alpha in various tissues including muscles were similar to each other.     

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.     

Cytosolic epoxide hydrolase from liver of control and clofibrate-treated mice. Structural comparison by HPLC peptide mapping

Cytosolic epoxide hydrolases purified from livers of control and clofibrate-induced male C57B1/6 mice were compared. The proteins were reduced, alkylated and cleaved with trypsin and chymotrypsin. The digests were analyzed by HPLC and no qualitative differences were observed in the peptide mapping profiles of the two types of epoxide hydrolase preparation. The amino acid compositions and N-terminal residues of selected tryptic peptides also gave identical results for the control and clofibrate-induced mice. Both intact proteins have e-amino-blocked N-termini. The two enzyme forms are concluded to have highly similar, if not identical, primary structures.Abbreviations HPLC high-performance liquid chromatography - DABITC dimethylaminoazobenzene isothiocyanate     

N-terminal amino acid sequence of two novel tumor-derived adenylate cyclase-stimulating proteins: identification of parathyroid hormone-like and parathyroid hormone-unlike domains

A 17,000 dalton human adenylate cyclase-stimulating protein has previously been purified from a human tumor associated with humoral hypercalcemia of malignancy. This report describes the purification of a related 7,000-9,000 MW protein from a second tumor, and provides N-terminal amino acid sequence of these two peptides. The sequences of the peptides are identical, defining the smaller peptide as an N-terminal portion of the larger peptide. The two peptides possess one region of strong homology with parathyroid hormone and a second divergent region. These structural similarities and differences may explain the similarities and differences which occur in patients with hyperparathyroidism and humoral hypercalcemia of malignancy.     

Sequence homology between the structural proteins of Kilham rat virus.

The capsid proteins of the autonomous parvovirus Kilham rat virus were purified and analyzed for peptide composition. Partial proteolysis mapping and two-dimensional thin-layer chromatography of tryptic peptide digests revealed extensive amino acid sequence homology between the two major Kilham rat virus capsid proteins.     

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.     

Homology between the glycoproteins of vesicular stomatitis virus and rabies virus

We compared the predicted amino acid sequences of the vesicular stomatitis virus and rabies virus glycoproteins by using a computer program which provides an optimal alignment and a statistical significance for the match. Highly significant homology between these two proteins was detected, including identical positioning of one glycosylation site. A significant homology between the predicted amino acid sequences of vesicular stomatitis virus and influenza virus matrix proteins was also found.     

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.     

Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium

An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene.     

Partial sequence data for the L-(+)-lactate dehydrogenase from Streptococcus cremoris US3 including the amino acid sequences around the single cysteine residue and at the N-terminus

The following amino acid sequence information has been determined for the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus cremoris US3: the C-terminal amino acid, the N-terminal sequence of the first 20 amino acids and the sequence of a 53-residue tryptic peptide containing the only cysteine residue in the protein. The enzyme was cleaved by alkali at the cysteine residue following reaction first with 5,5'-dithiobis(2-nitrobenzoic acid) and then with K14CN. This treatment yielded two cleavage products as well as some higher polymers and some uncleaved enzyme. The radioactive cleavage product was purified and its size indicated that the cysteine residue is 80 residues from the C-terminus. Comparisons of the sequences determined for the S. cremoris enzyme with those already known for dogfish lactate dehydrogenase indicate that the two enzymes are only distantly related since the sequence homology between them is limited and of borderline statistical significance.     

Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.     

Sequences of the soluble tryptic peptides from myoglobin of yellowfin tuna (Thunnus albacares).

1. Amino acid sequences of the soluble tryptic peptides of yellowfin tuna myoglobin, comprising 60% of the total residues, are presented. 2. The amino terminus is acetylated as shown by Fourier transform nuclear magnetic resonance spectroscopy of an N-terminal dipeptide. 3. Comparison of peptide sequences from yellowfin tuna myoglobin with corresponding regions of mammalian myoglobins shows obvious homology around the heme-attachment site and the carboxyl terminus, but marked dissimilarity is evident at other locations, such as the amino terminal region.     

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.     

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.     

The amino acid sequence around four cysteine residues in trout actin

Four unique carboxymethylcysteine-containing peptides were isolated from tryptic and chymotryptic digests of trout muscle actin carboxymethylated with iodo[2-(14)C]acetic acid in 6m-guanidinium chloride. The amino acid sequences of these peptides were determined and showed a high degree of homology with the corresponding sequences from rabbit actin. One of the radioactive peptides was the C-terminal peptide and another sequence probably contained the cysteine residue from the N-terminal region of the protein.     

N-terminal amino acid sequence of human urinary prokallikrein

The N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. (Formula; see text) The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-Gln-Ser-Arg(COOH). Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the alpha-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.     

Evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical

Bovine erythrocyte green heme binding protein and bovine erythrocyte flavin reductase have been isolated in highly purified forms and subjected to amino acid analysis and N-terminal amino acid sequence analysis. The two proteins possess similar amino acid compositions and identical N-terminal amino acid sequences. Moreover, the two proteins are immunochemically cross-reactive and are indistinguishable when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by double diffusion technique. This study provides evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.     

Structural evidence for an unblocked VHI sub-subgroup of human heavy chains.

The N-terminal amino acid sequence of an unblocked human heavy chain of an IgGK paraprotein from patient Tho is reported. This unblocked VH sequence belongs to the VHI subgroup and has striking structural homology to an unblocked VHI sequence previously reported for the IgGK paraprotein isolated from the serum of patient Bro. Direct sequence comparison of the Tho and Bro proteins reveals complete structural identity in 25 of the N-terminal 27 residues, with unique amino acid substitutions shared at the N-terminus and positions 16, 18, and 24. This remarkable sequence homology suggests that the two VH sequences represent examples of an unblocked VHI sub-subgroup. The Tho and Bro sequences possess an ala-glu-val basic triplet at positions 9 to 11 in common with the other previously reported VHI proteins which help to further establish this sequence triplet as a recognizable VHI subgroup-specific region. Moreover, in this regard, the unblocked sequences of Tho and Bro emphasize the importance of extending an unblocked VHI sequence beyond positions 9 to 11 before a subgroup assignment can be determined.