Murine hybridoma/plasmacytoma growth factor. Complete amino-acid sequence and relation to human interleukin-6

Murine interleukin-HP1 (HP1) was originally identified as a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas and plasmacytomas. This growth factor was recently shown to stimulate both normal B-cell differentiation and T-cell growth factor activity. We have determined the complete amino acid sequence of HP1 on 40 micrograms (approximately 2 nmol) protein using a combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC, peptide mapping and automated amino acid microsequence analysis. Ion-pairing chromatography was employed to isolate hydrophilic peptides which were not retained on conventional reversed-phase HPLC systems. The molecule consists of 187 amino acid residues with a calculated molecular mass of 21710 Da. Although there is virtually no similarity between the NH2-terminal region of HP1 and its human biological counterpart (26-kDa protein/interferon-beta 2 = B-cell stimulatory factor-2/interleukin-6), these studies demonstrate extensive amino acid similarity in the middle and COOH-terminal regions of these molecules suggesting that HP1 is the murine homologue of human interleukin-6.     

Cloning of cDNA for human T-cell replacing factor (interleukin-5) and comparison with the murine homologue.

We have cloned cDNA for T-cell replacing factor (interleukin-5), which replaces T-cell helper function for normal B cells which secrete immunoglobulin, from human T cell leukemia line, ATL-2, using mouse interleukin-5 cDNA as probe. Total nucleotide sequence of the cDNA (816 base pairs) was determined and compared with that of mouse interleukin-5 cDNA. The cloned cDNA encoded the interleukin-5 precursor of 134 amino acids containing an N-terminal signal sequence. Although the human interleukin-5 precursor is one amino acid longer than the murine homologue, the sizes of the mature proteins appear similar. The nucleotide and amino acid sequence homologies of the coding regions of human and murine interleukin-5 are 77% and 70%, respectively. Human interleukin-5 synthesized by the direction of the cloned cDNA induced immunoglobulin synthesis in human B cells stimulated by Staphylococcus aureus mitogen.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

NH2-terminal amino acid sequence of rabbit hepatopoietin A, a heparin-binding polypeptide growth factor for hepatocytes

Hepatopoietin A (HPTA) is an acidic heparin-binding polypeptide growth factor for hepatocytes with properties distinct from other known heparin-binding growth factors. HPTA is a heterodimer consisting of a heavy and a light polypeptide chain with Mr of 70,000 and 35,000 respectively. HPTA is a complete mitogen for hepatocytes in that it stimulates DNA synthesis in hepatocytes maintained in serum-free medium. Its complete purification from rabbit serum or human plasma was reported by us elsewhere (R. Zarnegar and G. Michalopoulos, 1989). In the present communication we report the N-terminal amino acid sequence of the HPTA light chain up to 24 residues (VVNGKPTRTNVGRMVSLKYRNKHI) and show that this sequence is unique and not related to any other proteins or growth factors based on computer search analysis. We have also raised antiserum against a synthetic peptide corresponding to the sequence of N-terminal amino acids residues 1 to 24, which recognizes the whole HPTA molecule. This antiserum as well as oligonucleotide probes corresponding to the N-terminal amino acid sequence of HPTA can be used as probes to identify tissue(s) of origin of this growth factor and assist in molecular cloning of its gene.     

Identification, cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.     

The structure of a naturally occurring 10K polypeptide derived from the amino terminus of bovine thyroglobulin

A combination of data derived from peptide sequencing and nucleic acid sequencing of cloned cDNA fragments has been used to define the complete amino acid sequence of a 10,000 M.W., thyroxine containing polypeptide derived from bovine thyroglobulin. This fragment, TG-F, which was obtained following reduction and alkylation, has been placed at the amino terminus of the parent protein with hormone located at residue 5 in the primary sequence of the thyroglobulin molecule. The carboxyl terminal sequence of this fragment -Cys-Gln-Leu-Gln is found on the N-terminal side of a lys residue, suggesting that the peptide bond cleavage which occurs to produce this 80 residue fragment from the parent (330K) thyroglobulin chain is a gln-lys. In addition, the amino acid sequence of this 10K fragment contains: No sequence which would be a substrate for glycosylation and no carbohydrate. Several repeated homologous amino acid sequences. A striking number of beta-bends predicted from Chou-Fasman analyses, particularly near its carboxyl terminus.     

The primary sequence of chicken myoglobin (Gallus gallus).

After enzymatic digestion of chicken myoglobin by trypsin, chymotrypsin or thermolysin, the separation of peptides was performed by column chromatography on various ion exchange resins. Each peptide was purified by high-voltage paper electrophoresis or by chromatography either on paper or on ion-exchange resin, and its complete amino acid sequence was then determined by the combined dansyl-Edman procedure and by endopeptidase digestions. The whole globin was submitted to automatic Edman degradation using the Beckman sequencer. Residues have been positioned from overlaps of sequence data between tryptic (T), chymotryptic (C) and thermolysin (Th) peptides. The stepwise degradation of the whole globin confirmed the alignment of the N-terminal third of the molecule. The combination of these different approaches has led to the complete determination of the 153 residues sequence forming the polypeptide chain of chicken myoglobin. Comparison of the established chicken myoglobin structure with those from other species shows a conservation of structure, although the avian protein exhibits more variations in its amino acid sequence than has been found between other known myoglobins which all belong to mammalian species.     

The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export.

flaFIX, the structural gene for the periplasmic P ring of the flagellar basal body of Salmonella typhimurium, was cloned. Two gene products with apparent molecular weights of 38,000 and 40,000 were identified by minicell analysis. Data from pulse-chase and membrane fractionation experiments and data on the inhibitory effect of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone all indicated that the 40-kilodalton protein was a precursor form which, after export across the cytoplasmic membrane accompanied by cleavage of a signal peptide, gave rise to the mature protein in the periplasm. The N-terminal amino acid sequence of the FlaFIX protein, predicted from the DNA sequence, conformed well to known signal peptide sequences. The results indicate that the P-ring protein of the basal body (unlike flagellin and possible some other external flagellar components) crosses the cytoplasmic membrane in a conventional signal peptide-dependent manner.     

Cloning and sequence analysis of the gene for glucodextranase from Arthrobacter globiformis T-3044 and expression in Escherichia coli cells

The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was cloned by using a combination of gene walking and probe methods and expressed on the recombinant plasmid pGD8, which was constructed with pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique open reading frame of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and 6 internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggested the enzyme was translated from mRNA as a secretory precursor with a signal peptide of 28 amino acids residues. The deduced amino acids sequence of the mature enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 daltons. The deduced sequence showed about 38% identity to that of the glucoamylase from Clostridium sp. G0005. The glucodextranase activity of transformant harboring pGD8 was about 40 mU/ml at 30 degrees C for a 16-h culture. Although the GDase that was produced from the transformant was shorter than authentic GDase by 2 amino acid residues at the N-terminal end side, its enzymatic properties were almost same as the authentic one. Two kinds of genes, dex1 and dex2, for endo-dextranases from A. globiformis T-3044 were also cloned into Escherichia coli cells. The N-terminal of the purified endo-dextranase from A. globiformis T-3044 agreed with the deduced amino acid sequence, after the 33rd alanine residue, of only the dex1 gene for edo-dextranase. This result suggests that the endo-dextranase is translated from mRNA as a secretory precursor with a signal peptide of 32 amino acids residues. The deduced sequence of endo-dextranase 1 and endo-dextranase 2 showed about 93% and 65% identity with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively.     

DNA sequencing of the eta gene coding for staphylococcal exfoliative toxin serotype A

We report the nucleotide sequence of a 1.45 kb segment containing the eta gene, coding for staphylococcal exfoliative toxin A (ETA), isolated from the recombinant plasmid pETA-J3. The coding region of 840 bp specified a polypeptide of 280 amino acid residues which included a putative 38 residue signal sequence. The amino acid composition deduced from the structural gene was in agreement with the results of peptide analysis of the ETA molecule reported by others. The sequence of the 35 N-terminal amino acid residues of ETA derived from Staphylococcus aureus strain ZM was also consistent with that deduced from the DNA sequencing.     

Structure and phylogeny of the crustacean hyperglycemic hormone and its precursor from a hydrothermal vent crustacean: the crab Bythograea thermydron

The structure of a well-known neurohormone involved in homeostasis regulation and stress response, the crustacean hyperglycemic hormone, was investigated in the deep-sea hydrothermal vent crab Bythograea thermydron. The neuropeptide was isolated from neurohemal organs (sinus glands) and its biological activity checked using an homologous bioassay. Partial amino acid sequence was established by a combination of Edman chemistry and mass spectrometry. Then, the sequence of the cDNA encoding the hormone precursor was determined. The preprohormone is composed of a 29 amino acid signal peptide, followed by a 41 amino acid associated peptide flanking the 72 amino acid hyperglycemic hormone. Comparison of these data with other known crab hyperglycemic hormone and prohormone sequences was performed using phylogenetic analysis methods.     

Evidence for production of a new lantibiotic (butyrivibriocin OR79A) by the ruminal anaerobe Butyrivibrio fibrisolvens OR79: characterization of the structural gene encoding butyrivibriocin OR79A

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79A was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5' region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.     

Structural characterization of a murine myeloid leukaemia inhibitory factor

A leukaemia inhibitory factor (LIF) which induces macrophage differentiation in M1 murine myeloid leukaemia cells and suppresses their proliferation in vitro has been isolated in sufficient quantities (30 micrograms) from Krebs ascites tumour cell conditioned medium to permit its partial characterization by amino acid sequence analysis. The combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC technology and microsequence analysis has enabled the positive identification of 125 of the total 179 amino acid residues (70%) in the molecule. The amino acid sequence data reported here permitted the isolation of a partial cDNA clone encoding LIF [Gearing et al. (1987) EMBO J. 6, 3995-4002]. A candidate C-terminus of the LIF molecule predicted from the amino acid sequence was confirmed by subsequent isolation of a cDNA clone corresponding to the C-terminus of the protein. No strong similarity was revealed when the amino acid sequence of LIF was compared with other haemopoietic growth factors, in particular granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and tumour necrosis factor-alpha or interleukins. The protein sequence data reported here indicate three sites of post-translational modification (N-linked glycosylation).     

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.     

Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans

A novel serine protease, named as scolonase, was purified and characterized from the tissue of the Korean centipede, Scolopendra subspinipes mutilans. Purified scolonase showed an apparent molecular weight of 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and an isoelectric point of 4.8 on isoelectric focusing gel. Scolonase was able to preferentially hydrolyze arginine over lysine at the cleavage site among the several synthetic peptide substrates. Scolonase has also a potent fibrinolytic activity by converting human Glu-plasminogen to activated plasmin due to the specific cleavage of the molecule at the peptide bond Arg(561)-Val(562). The enzyme activity of scolonase was completely inhibited by phenylmethanesulfonyl fluoride and difluorophosphate. The cDNA encoding scolonase was cloned from the cDNA library of the centipede constructed with oligonucleotide probe, which was designed on the basis of the N-terminal amino acid sequence of scolonase. The deduced complete amino acid sequence of scolonase demonstrated that the protein is composed of 277 amino acids including 33 amino acids as a leader sequence, and that it has significant sequence homology with other serine proteases.     

Phospholamban: dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms

Structural characterization of a 40 amino acid peptide with high intrinsic growth hormone releasing activity isolated from a human pancreatic tumor which had caused acromegaly was accomplished by gas phase sequence analyses of the intact peptide and its carboxy terminal cyanogen bromide digestion fragment. High pressure liquid chromatography of the native peptide and synthetic replicates showed that the molecule possessed a free acid rather than an amidated carboxy terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly- Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-OH using 1.8 nmoles of material. The structural identity of this material with a previously characterized fragment of a larger growth hormone releasing peptide isolated from a different human tumor is discussed.     

Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

Molecular cloning of rat glucose-dependent insulinotropic peptide (GIP).

A cDNA clone encoding glucose-dependent insulinotropic peptide (GIP) was identified that consisted of 34 bp of 5' untranslated sequence, an open reading frame of 432 bp and 115 bp in the 3' untranslated region. The deduced amino acid sequence revealed a 144 amino acid preprohormone consisting of a 43 amino acid N-terminal extension including a signal peptide, a 42 amino acid hormone, and a 59 amino acid C-terminal extension. Rat GIP differs from the human hormone by two amino acid substitutions: arginine for histidine at position 18 and leucine for isoleucine at position 40. A single mRNA from small intestine of approximately 800 bases was identified on Northern blot analysis in equivalent amounts in proximal and distal small intestine.     

Phospholipid transfer protein: full-length cDNA and amino acid sequence in maize. Amino acid sequence homologies between plant phospholipid transfer proteins

We have determined the primary structure of a phospholipid transfer protein (PLTP) isolated from maize seeds. This protein consists of 93 amino acids and shows internal homology originating in the repetition of (do)decapeptides. By using antibodies against maize PLTP, we have isolated from a cDNA library one positive clone (6B6) which corresponds to the incomplete nucleotide sequence. Another cDNA clone (9C2) was obtained by screening a size-selected library with 6B6. Clone 9C2 (822 base pairs) corresponds to the full-length cDNA of the phospholipid-transfer protein whose mRNA contains 0.8 kilobase. Southern blot analysis shows that the maize genome may contain several PLTP genes. In addition, the deduced amino acid sequence of clone 9C2 reveals the presence of a signal peptide. The significance of this signal peptide (27 amino acids) might be related to the function of the phospholipid-transfer protein. The amino acid sequence of maize PLTP was compared to those isolated from spinach leaves or castor bean seeds which exhibit physicochemical properties close to those of the maize protein. A high homology was observed between the three sequences. Three domains can be distinguished: a highly charged central core (around 40-60), a very hydrophobic N-terminal sequence characteristic of polypeptide-membrane interaction, and a hydrophilic C terminus. A model for plant phospholipid-transfer proteins is proposed in which the phospholipid molecule is embedded within the protein with its polar moiety interacting with the central hydrophilic core of the protein, whereas the N-terminal region plunges within the membrane in the transfer process.