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A novel family C of the genes that encode bombyxin, an insulin-related brain secretory peptide of the silkmoth Bombyx mori: Isolation and characterization of gene C-1

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Abstract A novel gene C-1 encoding bombyxin, an insulin-family peptide which is produced by the brain neurosecretory cells of the silkmoth Bombyx mori, has been cloned and characterized. The nucleotide sequence of this gene and the deduced amino acid
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  Insect Biochem. Vol. 20, No. 3, pp. 295-303, 1990 0020-1790/90 3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1990 Pergamon Press pie A NOVEL FAMILY C OF THE GENES THAT ENCODE BOMBYXIN AN INSULIN-RELATED BRAIN SECRETORY PEPTIDE OF THE SILKMOTH BOMBYX MORI ISOLATION AND CHARACTERIZATION OF GENE C-1 MASAFUMI WAMI '*, TAKASHI ADACHI , HIDEHIKO KONDO 1, ATSUSHI KAWAKAMI , YOSHIAKI SUZUKI , HIROMICHI NAGASAWA , AKINORI SUZUKI and HIRONORI SI-IIZAKI tDepartment of Biology, School of Science, Nagoya University, Nagoya 464-01, 2National Institute for Basic Biology, Okazaki 444 and 3Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Tokyo 113, Japan Received 18 September 1989; revised and accepted 15 November 1989) Abstraet--A novel gene C-1 encoding bombyxin, an insulin-family peptide which is produced by the brain neurosecretory cells of the silkmoth Bombyx mori, has been cloned and characterized. The nucleotide sequence of this gene and the deduced amino acid sequence are definitely different from those previously characterized for the A- and B-family bombyxin genes. Southern hybridization indicates that the Bombyx genome contains multiple gene copies closely related to this gene comprising a novel family C. Tertiary structural and functional features of bombyxins are speculated from their primary structure. Inspection of the sequences of the bombyxin genes so far characterized suggests that they may be the functional processed genes. Key Word Index: Bombyx mori, bombyxin, brain neurosecretory hormone, processed gene, insulin INTRODUCTION Bombyxin is a 5 kDa brain secretory peptide of the silkmoth Bombyx mori which shows amino acid sequence homology with insulin-family peptides (Nagasawa et al., 1986; Ishizaki and Suzuki, 1988; Suzuki et al., 1989). This peptide can cause resump- tion of adult development when injected into brain- removed dormant pupae of the saturniid moth Samia cynthia ricini (Ishizaki and Ichikawa, 1967) by stimu- lating the prothoracic glands to synthesize and release eedysone necessary for molting and metamorphosis (Nagasawa et al., 1984a). Purification of bombyxin from Bombyx heads revealed the presence of highly heterogeneous molecular species, the amino acid sequences of which varied slightly from one another (Nagawawa et al., 1984b, 1986, 1988). The primary structure of native bombyxins has been determined completely for bombyxin-IIs (Nagasawa et al., 1986, 1988) and -IV (Maruyama et al., 1988), and partially for bombyxin-I, -III and -V (Nagasawa et al., 1984b; Jhoti et al., 1987). They are the heterodimeric molecule consisting of the A- and B-chains which are connected together by disulfide bonds in exactly the same way as in insulin (Nagasawa et al., 1988) to form an insulin-like tertiary structure (Jhoti et al., 1987). Despite its clear-cut prothoracicotropic activity on Samia, the physiological function of bombyxin in Bombyx itself has not yet been clarified but its insulin-like structure is thought to suggest some essential functions in Bombyx growth and/or metabolism. *Author to whom all correspondence should be addressed. In the previous study we isolated a bombyxin gene from the Bombyx genome (Iwami et al., 1989). Sub- sequently, we characterized a bombyxin gene cluster composed of a tandem repeat of paired genes (Kawakami et aL, 1990) and a still larger number of bombyxin gene copies has been suggested to exist in the Bombyx genome (Kawakami et al., 1990; Adachi et al., 1989). Structural comparison of these genes permitted us to classify the bombyxin genes into two groups, the A- and B-familes. Thus, bombyxin genes A-I, A-2, A-3, B-I and B-2 have been defined (Kawakami et al., 1990). All of these bombyxin genes have the same domain organization as in insulin genes (Chan et al., 1981; Steiner et al., 1985) to encode signal peptide/B-chain/C-peptide/A-chain, but differ from them in having no introns. Two distinct cDNAs encoding bombyxin have also been isolated from a Bombyx larval brain eDNA library (Adachi et aL, 1989). One of these (2Bb360) has apparently been derived from the gene A-2 transcript (Kawakami et aL, 1990), while the other (2Bb204) encodes a bombyxin which has not been identified previously. Since this 2 Bb204 eDNA was of partial length to allow us to predict the amino acid sequence of only the A-chain and C-peptide, it was desired to clarify the complete sequence of this bom- byxin. To this end we tried to clone a gene encoding this new bombyxin. We describe here the character- ization of a novel bombyxin gene thus cloned. The amino acid sequence of the bombyxin deduced from this gene is significantly different from the A- and B-family members and all mature bombyxins so far characterized. In addition, Southern hybridization analysis indicates that a group of genes closely related to this gene is present in the Bombyx genome. We 295  296 MASAFUMI WAMI et al. therefore define this bombyxin gene group as the C-family and designate the presently isolated gene as gene C-1. We discuss the structural features of the C-I gene with those of others previously character- ized. The view that bombyxin genes are the processed gene is presented. MATERIALS AND METHODS Isolation of bombyxin clones High molecular weight DNA from the whole body of newly hatched first-instar of Bombyx mori (Kinshu X Showa) was partially digested by Sau3AI in the range of 10-23 kilo base-pairs (kb). The fragments were dephospho- rylated and inserted into the BamHI arms of 2EMBL3 (Frischauf et al. 1983). The library was packaged in vitro and screened at a stringent hybridization condition with the 32p-labeled bombyxin eDNA insert in clone ~.Bb204 (Adachi et al. 1989). The hybridization was carried out at 65°C in 6X (SSC: 0.15 M NaCI 0.015 M Na-citrate), 10 x Denhardt (Denhardt: 0.02 bovine serum albumin/ 0.02 Ficoll/0.02 polyvinylpyrrolidone), 0.25 mg/ml sonicated salmon sperm DNA and oligolabeled probe DNA (Feinberg and Vogelstein, 1983). The filters were washed at 65°C in succession with the following solutions: 2 x SSC and 0.1 SDS for 2h; 0.2 x SSC and 0.1 SDS for I h. Positive phage were picked up and amplified by plate lysis. One of the positive phages, 24K403, was further analyzed. Sequencing analysis The HindlII/KpnI fragment hybridized with the bom- byxin eDNA probe was subeloned into pUC vectors (Yanisch-Perron et al. 1985). The done was sequenced on both strands by the chain-termination method (Sanger et al. 1977) with deoxy-7-deaza-guanosine triphosphate and [~(-'S]dCTP (Mizusawa et aL 1986). Sequences were ana- lyzed using computer programs (DNASIS, Hitachi Software Engineering). Southern hybridization analysis Genomic DNA from one pair of silk glands of Bombyx (J122 x Cl15) was digested by restriction enzymes and quantified after phenol exraction and ethanol precipitation. The digested DNA (5.8/~g for each lane) was electro- phoresed on a 0.7 agarose gel and transferred to a nylon membrane (Biodyne, Pall). The blot was hybridized at 42°C with the 32p-labeled bombyxin eDNA probe in the solution of 50 formamide, 5X SSC, 5X Denhardt, 50 mM phos- phate buffer (pH 5.95), 0.25 mg/ml sonicated salmon testis DNA. Washing was done at 65°C in SSC and 0.1 SDS for more than 3 h. The washed membranes were exposed to X-ray films at -70°C with intensifying screens. RESULTS Structure of bombyxin C- I gene The nucleotide sequence of bombyxin C-1 carried by clone 24K403 is shown in Fig. 1. The open reading 5 -AAGCTTCCATATAAAATGAGAGACTTTCGCACGCTCG~ 46 ~ MetLysLeu -17 CAT ,~TGCCATCACAACGAGTAGCACGTGAGGAATCATCATGAAACTG 94 SIGNAL PEPTIDE 10 1l ValMetLeuLeuValValValSerAlaMetLeuValLeuGlyGlyAla i GTCATGCTCCTTGTTGTCGTTTCTGCCATGCTCGTGTTAGGAGGAGCA 142 B-CHAIN I I i0 GlnThrAlaSerGlnPheTyrCysGlyAspPheLeuAlaArgThrMet 16 CAAACAGCCAGCCAATTTTATTGTGGAGACTTTTTGGCGCGTACAATG 190 o 30 SerSerLeuCysTrpSerAspMetGlnLysArgSerGlySerGlnTyr 32 TCCAGCCTGTGTTGGTCGGACATGCAGAAACGAAGCGGATCCCAGTAC 238 C PEPTIDE 40 AlaGlyTyrGlyTrpProTrpLeuProProPheSerSerSerArgGly 48 GCGGGCTACGGCTGGCCGTGGCTGCCCCCCTTCTCCTCGTCTCGTGGG 286 A-CHAIN 5@ 6O LysArgGlylleValAspGluCysCysTyrArgProCysThrIleAsp 64 AAACGCGGTATTGTCGATGAGTGCTGCTACAGACCCTGCACAATAGAC 334 70 I ValLeuMetSerTyrCysAspAsn*~ 72 GTTCTGATGTCATACTGCGATAACTAGTGCGGAAAACTCGACAATTTG 382 ACAATTTTCGTAAACGCCTGATTTATATTAAATAAATTATGTACTAAA 430 AT~ ~-~GTTTTATTAATGGTACC-3 456 Fig. 1. Bombyxin C-I gene sequence with deduced amino acid sequence. Nucleotides are numbered sequentially from the HindIII site at the Y-end. The amino acid sequence is numbered sequentially from the presumed N-terminus of bombyxin C-1 B-chain. Signal peptide, B-chain, C-peptide and A-chain are bracketed. Asterisks indicate termination codons. Direct repeats are boxed, A putative transcriptional initiation sequence and a polyadenylation signal sequence are indicated by thick and thin underlines, respectively.  Bombyxin C-family gene 297 frame of 273 nucleotides apparently encodes a pre- cursor molecule for bombyxin, preprobombyxin, that consists of the signal peptide, B-chain, C-peptide and A-chain in this order. This domain organization is the same as that of the bombyxin genes and cDNAs previously characterized (Iwami et aL 1989; Kawakami et al. 1989; Adachi et aL 1989). The 3 -part (position 228-431) of the gene C-1 sequence is identical to a partial-length cDNA, 2Bb204 (Adachi et al. 1989), indicating that this cDNA has been derived from the transcript of gene C-1. We find three potential initiation codons (Met -7, -15 and -19) and a termination codon appears at four frames upstream of Met -19. Considering general features for the signal peptide (Watson, 1984) and the consensus sequence for translation start site in Drosophila (Cavener, 1987), we suspect that Met -19 may be the initiator. Gin 1 may represent the N- terminal amino acid residue of the B-chain, because the Gin residue is most probably converted post- translationally to pyroglutamate (Turner, 1986), the conserved N-terminal residue of the B-chain of the native bombyxins (Nagasawa et al. 1984b, 1986, 1988). There are paired basic amino acids (Lys-Arg) at both termini of the C-peptide like in other preprobombyxins already characterized (Iwami et al. 1989; Kawakami et al. 1990; Adachi et al. 1989), suggesting that the post-translational processing occurs at these sites to generate a mature bombyxin through excision of the C-peptide by a trypsin- like endopeptidase. A putative transcriptional initi- ation sequence (CACATCC; Kaswakami et al. 1989) and a polyadenylation signal sequence (AATAAA) are observed at 41 bp upstream from the translation initiation site and at 54 bp downstream from the termination site, respectively, suggesting that this gene is transcribed by RNA polymerase II. The coding region of gene C-1 apparently lacks introns and no splicing signals are found in the untranslated regions. Thus the bombyxin C-1 gene has no introns as in the case of other bombyxin genes previously characterized (Iwami et al. 1989; Kawakami et al. 1989). It is worth noting that the C-1 gene has a direct repeat (AATAT-T) flanking the both sides of the presumed transcribed region, though the repeat is incomplete (Fig. 1, boxed). The presence of a direct repeat together with a lack of introns in gene C-I, and in other bombyxins also, provides evidence for arguing that bombyxin genes are the processed gene, as is treated in detail in the Discussion. Nucleotide sequence comparison of C-1 with other bombyxin genes The nucleotide sequence of the C-1 gene is com- pared with the A- and B-family genes by Harr-plot analysis (Fig. 2). Gene C-1 shares a high homology with the A-family genes only in the C-peptide and A-chain regions as illustrated in Fig. 2(b), in which C-1 is compared with A-1 as a representative of the A-family genes. Similarly, C-1 shares significant homology with the B-family genes only at the C- peptide and A-chain regions [Fig. 2(c)]. By contrast, when the A- and B-families are compared, high homology is seen not only in the C-peptide and A-chain but also in the B-chain [Fig. 2(a)]. These results indicate that C-1 is related more distantly to the A- and B-family genes than the A- and B-families are related to each other. The evolutionary distance per nucleotide site is calculated according to Kimura (1981). The B- chain of C-I is definitely distant from that of others (Table 1), substantiating the notion that C-1 is evolutionarily remote from the A- and B-families. Amino acid sequence comparison of C-I with other bombyxins The amino acid sequence of preprobombyxin de- duced from gene C-1 is aligned to the sequences deduced from the A- and B-family genes and those of the native bombyxins (Fig. 3). Obviously the C-I sequence is considerably different from other se- quences. The A-chain of C-1 bombyxin has 22 amino acid residues, being longer at the C-terminus than the A-chain of all other bombyxins. Four residues of the A-chain are substituted when bombyxin C-1 is com- pared with A-family bombyxins, while 5 residues of bombyxin C-I A-chain differ from the A-chains of the B-family members. In contrast, the A-chains are a b c \ I .ab \ N • ¢5 N \ \ IslBIc AI IslBIClAI Islelc Ai-- B--1 A--1 B--1 Fig. 2. Homology Harr-plot analysis of nucleotide sequences. (a) Bombyxin A-I (Iwami et al. 1989) vs B-I (Kawakami et al. 1989), (b) bombyxin C-1 vs A-l, (c) bombyxin C-1 vs B-I. S, B, C and A in boxes represent signal peptide, B-chain, C-peptide and A-chain regions, respectively. Computer program (DNASIS) was used for processing of nucleotide sequence data. Check size, 15; number of match bases, 11.  298 MASAFUM WAM t al. Table 1. Evolutionary distance per nucleotide site (K)* Chain or peptide Comparison A B C Bombyxin A-I vs B-I 0.47 0.39 0.64 A-I vs C-I 0.40 0.75 0.54 B-l vs C-l 0.40 1.02 0.56 Human insulin vs rat insulin-I 0.07 0.20 0.44 *K values were calculated using the 3ST model (Kimura, 1981). Nucleotide sequences were referred to from lwami t al. (1989) for bombyxin A-l, Kawakami et al. (1989) for bombyxin B-l, lwami et al. (1990) for bombyxin C-l, Bell et al. (1980) for human insulin and Lomedico t al. (1979) for rat insulin-I. completely conserved between the members within each of the A- and B-families. When the B-chain is compared, the difference of C-1 bombyxin from the others is more prominent. In addition to the deletions and insertions of some amino acids, C-1 deviates from the A- and B-family members sharing only 44 and 36% homology, respectively. These values are lower than the homology between the A- and B- families (61%). Remarkably, 4 residues of the B- chain at positions 9, 10, 16 and 24 are substituted compared to the A- and B-families in which these 4 sites are completely conserved. The difference of C-1 from bombyxin-III and -IV is also prominent. These comparisons thus clearly indicate that bom- byxin C-1 is definitely distinct from any other known bombyxins. Southern hybridization analysis Southern hybridization using the bombyxin C-1 gene as a probe under a stringent condition in which this probe did not hybridize with the A- and B-family members detected two hybridization signals in the Bam HI- and the HindIII-digested genomic DNAs and three signals in the EcoRI-digested DNA (Fig. 4). Since the 4.4kb signal in the BamHI lane contained a single C-1 gene presently characterized (details for restriction analysis will be given else- where), we consider that the Bombyx genome con- tains at least two, possibly three, gene copies closely related to C-1. This fact has led us to consider that gene C-1 and others closely related to it comprise a novel bombyxin gene family, C-family, DISCUSSION C-family a novel bombyxin gene family From the nucleotide and amino acid sequence comparisons and the detection of closely related multiple gene copies by Southern hybridization analyses of the Bombyx genomic DNA, we defined in this paper a novel bombyxin C-family to which the I A-I bombyxin A-family A-2 A-3 II B-family [ B-I B-2 C-family C-I others IV human insulin human relaxin SIGNAL PEPTIDE 1 i0 20 I MKI LLAI AL MLSTVMWVST MMKTSVMFMLV IVISLMCSGEA ........... F ....... S NALWMRLLPLLALLALWGPDPAAA MPL FLFHLLEFCLLLNQFSRAVAA 0 B-CHAIN I I0 20 QQPQRVHTYCGRHLARTLADLCWEAGVD .... E ........... N ..... E--- G .............. N ........ >Q---A ...................... QE VARTYCGRHLADTLADLC FGVE >QEANVAHHYCGqrlrLANTL][DLCMDTSVE 3O FVNQHLCGSHLVEALYLVCGERGFFYTPXT KWKDDVIKLCGRELVRAQIAICGMSTMSKRSL O00 O0 O0 C-PEPTIDE A-CHAIN l I A-I [R bombyxin A-family A-2 -- A-3 -- II I B-1 KR B-family B-2 -- I C-family C-I KR others III IV human insulin human relaxin 10 20 30 i i0 20 V I ~ T GAQFASYGSAM LMPY SEGRGKR GIVDECCLRPCSVDVLLSYC D ............... A ....................... -D--YV .......... A ........................ GGAQYAPY FW TRQY LGSRGKR GVVDECCFRPCTLDVLLSYCG S ......................................... S@¥A~Y(~M 10~ ~SRGKR GIVDECC~PC~VL~YC~ GVVDECCLqPCT~DVV TTC GVVDECCIQPCTLDVLATYC GIVBQCCTSICSLYQLENYCN 95 amino acids) QKKR RPYVALFELCCLIGCTKRSLALYC • O00 O0 O O000 0 O0000 OO0 O0 O0 RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR SQEDA IO Fig. 3. Amino acid sequence comparison between preprobombyxins (Iwami et al. 1989; Kawakami et al. 1989), bombyxins (Nagasawa et al. 1984b, 1986; Maruyama et al. 1988), human insulin (Bell et al. 1980) and human relaxin (Hudson et al. 1983). Gaps are introduced for maximum alignment. Amino acids are numbered from the N-terminus of each domain of insulin. The residues unique to preprobombyxin C-I compared to other preprobombyxins and bombyxins are boxed. Boxes with thick sides, in particular, are applied when the residues are completely conserved except for preprobombyxin C-I. The residues which are completely conserved throughout the peptides shown and those conserved among bombyxins are indicated by the open and solid circles, respectively. < Q, pyroglutamate.  BomH EcoR I Hind m F M F M F M 23 9 4 6.7 4 4 2.3 2.0 |kb) Fig. 4. Southern hybridization of the Bombyx genomic DNA. Restriction enzymes used for the genomic DNA digestion are indicated above the autoradiograms kb shown on the left were estimated from a size marker ;tDNA digested by HindlII F female; M male. No sexual difference was observed in the band pattern and intensity. 299
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