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.     

Primary structure of a member of the serpin superfamily of proteinase inhibitors from an insect, Manduca sexta

A cDNA clone isolated from a fat body cDNA library from an insect, Manduca sexta, has been sequenced and shown to code for a member of the serpin family of proteinase inhibitors. The cDNA has an open reading frame which codes for a 392-residue polypeptide of Mr = 43,500 with a hydrophobic NH2-terminal sequence which appears to be a signal peptide. An alignment of this amino acid sequence with 11 members of the serpin superfamily reveals that the insect protein is 25-30% identical with most members of the superfamily. The alignment was used to construct an evolutionary tree of the serpin sequences analyzed, which indicates that the progenitor of the M. sexta serpin and the human serpins most closely related to it diverged from other serpin genes prior to the divergence of the vertebrates and invertebrates. The M. sexta serpin is predicted to inhibit elastase due to the presence of alanine at the P1 position of its reactive center and is classified as an alaserpin. A glycoprotein of Mr = 47,000 isolated from hemolymph of M. sexta larvae has an NH2-terminal sequence identical to that deduced from the alaserpin cDNA clone and inhibits porcine pancreatic elastase and bovine chymotrypsin.     

Molecular cloning and characterization of a transferrin cDNA from the white-spotted flower chafer, Protaetia brevitarsis.

A full-length cDNA clone with high homology to insect transferrin genes was cloned by screening a Protaetia brevitarsis cDNA library. This gene (PbTf) had a total length of 2338 bp with an open reading frame (ORF) of 2163 bp, and encoded a predicted peptide of 721 amino acid residues. Like known cockroach, termite, and beetle transferrins, PbTf appears to have residues comprising iron-binding sites in both N- and C-terminal lobes. The deduced amino acid sequence of the PbTf cDNA was closest in structure to the beetle Apriona germari transferrin (68% protein sequence identity). Northern blot analysis revealed that PbTf exhibited fat body-specific expression and was upregulated by wounding, bacterial or fungal infection and iron overload, suggesting a functional role for PbTf in defense and stress responses.     

The nucleotide sequence of rabbit liver transferrin cDNA

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.     

Molecular cloning and analysis of residues associated with iron binding of Spodoptera litura transferrin

We isolated transferrin cDNA from tobacco cutworm (Spodoptera litura) and refer to it as SlTf (Spodoptera litura transferrin). The 2,237-bp SlTf cDNA encoded 685 amino acids, with a predicted Mw of 76.3 kDa and an isoelectric point of pH 7.97. The amino acid sequence of the SlTf protein had 11?C81% similarity with those of other reported animal transferrins, showing the highest similarity with another Lepidopteran insect, the silkworm (Bombyx mori), and the lowest similarity with atlantic cod (Gadus morhua) serum transferrin. Phylogenetic tree analysis showed that SlTf was close to transferrins of B. mori and M. sexta. By urea-polyacrylamide gel electrophoresis, four different iron-bound forms (apo-, C-terminal monoferric, N-terminal monoferric and diferric) were found from both SlTf and human transferrin, suggesting the C-lobe iron-binding motif of SlTf possesses the iron-biding activity, although its amino acid sequence is not well conserved compare to that of vertebrate transferrins. Accordingly, we suggest that the amino acid residues of iron-binding sites in SlTf are different from those of human serum transferrin, however the iron-binding capacity is conserved in both the C-lobe and the N-lobe of SlTf.     

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.     

Drosophila melanogaster transferrin. Cloning, deduced protein sequence, expression during the life cycle, gene localization and up-regulation on bacterial infection.

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks iron-binding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.     

Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.     

Isolation and characterization of the iron-binding properties of a primitive monolobal transferrin from Ciona intestinalis

Transferrins are bilobal glycoproteins responsible for iron binding, transport, and delivery in many higher organisms. The two homologous lobes of transferrins are thought to have evolved by gene duplication of an ancestral monolobal form. In the present study, a 37.7-kDa primitive monolobal transferrin (nicatransferrin, or nicaTf) from the serum of the model ascidian species Ciona intestinalis was isolated by using an immobilized iron-affinity column and characterized by using mass spectrometry and N-terminal sequencing. The protein binds one equivalent of iron(III) and exhibits an electron paramagnetic resonance spectrum that is anion-dependent. The UV/vis spectrum of nicaTf has a shoulder at 330 nm in both the iron-depleted and the iron-replete forms, but does not display the approximately 460 nm tyrosine-to-iron charge transfer band common to vertebrate serum transferrins under the conditions investigated. This result suggests that iron may adopt a different binding mode in nicaTf compared with the more highly evolved transferrin proteins. This difference in binding mode could have implications for the physiological role of the protein in the ascidian. The genome of C. intestinalis has genes for both a monolobal and a bilobal transferrin, and the sequences of both proteins are discussed in light of the known features of vertebrate serum transferrins as well as other transferrin homologs.     

Molecular Cloning and Characterization of Transferrin from Wax Moth, Galleria mellonella

Complete mRNA sequence of transferrin from Galleria mellonella was obtained, and compared with those of other species. Until now, two types of insect transferrin were reported. Transferrins in cockroach and termite have two iron binding sites while those in most other insect groups, studied for the protein, have only one. It was suggested that the presence of two types of transferrin was related with transferrin evolution, because vertebrate transferrins have two iron binding sites, called N and C terminal lobe. It was shown that G. mellonella transferrin also has only one iron binding site (N terminal lobe), and the deduced amino acid sequence was most similar to those of Manduca sexta and Bombyx mori.     

Cloning and expression of 32 kDa ferritin from Galleria mellonella

We have sequenced a cDNA clone encoding 32-kDa ferritin subunit in the Wax Moth, Galleria mellonella. The 32-kDa ferritin subunit cDNA was obtained from PCR using identical primer designed from highly conserved regions of insect ferritins. RACE PCR was used to obtain the complete protein coding sequence. The 32-kDa ferritin subunit encoded a 232 amino acid polypeptide, containing a 19 leader peptide. The iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region of the wax moth 32-kDa ferritin subunit mRNA. The 32-kDa sequence alignment had 78 and 69% identity with Manduca sexta and Calpodes ethlius (G), respectively. The G. mellonella ferritin subunits showed minimal identity with each other (19%). The glycosylation site (Asn-X-Ser/Thr) was found in the 32-kDa subunit but not in the 26-kDa subunit. Northern blot analysis showed that the mRNA expression of the 32-kDa ferritin was detected in the fat body and midgut. The fat body expression increased after 6 h and the mRNA in midgut dramatically increased about 3-fold the expression level at 12 h after iron feeding. Western blot revealed that a protein level of the 32-kDa subunit is abundant in midgut after 12 and 24 h iron feeding.     

cDNA cloning and complete primary structure of the small, active subunit of human carboxypeptidase N (kininase 1)

The human plasma metallo-protease carboxypeptidase N of Mr 280,000 consists of two small, enzymatically active subunits of Mr 50,000 and two large subunits. Only the large subunits are glycosylated. They may have a function in stabilizing the complex in plasma. The N-terminal sequence of the small subunit was determined from the isolated protein and used to specify a unique 59-mer oligonucleotide probe. A cDNA clone of 1.7 kbp containing the entire coding sequence of the small subunit of carboxypeptidase N was isolated from a human-liver cDNA library. The cDNA clone encodes a signal sequence of 20 amino acids and the 438 amino acids of the mature subunit. There is a remarkable primary structure similarity of 49% to bovine carboxypeptidase E (enkephalin convertase). A more distant relationship to the bovine pancreatic, digestive carboxypeptidases A and B or even to the metallo-endopeptidases is based mainly on the occurrence of conserved, mechanistically important residues.     

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.     

Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.     

Molecular cloning and characterization of a cDNA encoding a transferrin homolog from Bombyx mori

We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori. The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.     

cDNA cloning and deduced amino acid sequence of microvitellogenin, a female specific hemolymph and egg protein from the tobacco hornworm, Manduca sexta

Microvitellogenin is a female-specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly(A)+ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by using antiserum against microvitellogenin. Northern blot analysis of poly(A)+ RNA isolated from different life stages and sexes reveals that mRNA coding for microvitellogenin is only present in adult female fat body. Immunoprecipitation of the protein product translated from hybrid selected mRNA indicates that the cDNA clone is specific for microvitellogenin. The complete nucleotide sequence of the 834-base pair cDNA insert has been determined by the dideoxy chain termination method. The cDNA sequence predicts that microvitellogenin is a protein of 232 residues with a calculated molecular weight of 26,201. The cDNA also predicts an amino-terminal extension of 17 residues which are not present in the mature form. This sequence appears to be a signal peptide. A comparison of the translated amino acid sequence with the sequences in the National Biomedical Foundation protein library did not establish any sequence homology with other known proteins.     

Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane

Peripherin, a 39-kDa membrane protein, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane by use of monoclonal antibodies and immunocytochemical labeling techniques. As an initial step in determining the structure and function of this protein, we have cloned and sequenced cDNA containing its complete coding sequence. A bovine retinal lambda gt11 expression library was screened with the antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent lambda gt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity-purified peripherin are in agreement with the cDNA sequence. The cDNA sequence predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine-linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The possible function of peripherin in relation to its primary structure is discussed.     

A bacteria-induced, intracellular serpin in granular hemocytes of Manduca sexta.

Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.