©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Precursors of the Bee Venom Constituents Apamin and MCD Peptide Are Encoded by Two Genes in Tandem Which Share the Same 3`-Exon (*)

Michael Gmachl (§) , Günther Kreil

From the (1) Institute of Molecular Biology, Austrian Academy of Sciences, A-5020 Salzburg, Austria

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

From a cDNA library prepared from venom glands of worker bees, clones encoding the precursors of apamin and MCD peptide have been isolated. The cDNAs are similar at the 5`-ends and identical in their 3`-regions. Analysis of the corresponding genes has revealed the existence of six exons separated by introns rich in A + T. Starting from the 5`-end, these exons are arranged in the following order: three exons of the mast cell-degranulating (MCD) peptide precursor, two exons of the gene for the apamin precursor, and finally a 3`-exon present in both cDNAs. This suggests that the bulk of the apamin gene resides in the third intron of the MCD peptide gene. Using inverse polymerase chain reaction, a segment of genomic DNA upstream of the first exon of the MCD precursor gene was obtained. The sequence of this segment shows 81% identity to the DNA sequence preceding the first exon of the apamin gene and both contain a putative TATA box. We thus propose that the mRNA encoding the apamin precursor originates from a primary transcript which starts in the third intron of the MCD peptide gene. Both cDNAs encode unusually small precursors comprising only 46 amino acids in case of apamin and 50 in the case of the MCD peptide.


INTRODUCTION

The venom from the gland of the honeybee, Apis mellifera, produces an aqueous secretion which contains in significant quantity only two enzymes and four peptides (1, 2) . The enzymes are a phospholipase A and a hyaluronidase, and the amino acid sequence of these proteins has been determined (3, 4, 5, 6) . The main constituent of bee venom is melittin which represents 50-60% of the dry weight. This is a lytic peptide that forms an amphipathic helix (7) and inserts into phospholipid bilayers. Other peptides, each present at less than one-tenth the amount of melittin, are mast cell-degranulating (hence the name MCD)() peptide, apamin, and secapin. MCD peptide, also called peptide 401 (8) contains 22 amino acids (see Fig. 1). It has a potent histamine-releasing activity (1, 2) , and it also acts as an anti-inflammatory agent (8) . Upon injection into the brain, MCD peptide elicits in a dose-dependent manner a variety of symptoms ranging from arousal to convulsions (9) . It was shown subsequently that in rat brain MCD peptide binds with high affinity to voltage-sensitive potassium channels (10, 11, 12) . Apamin, which contains 18 amino acids and shows some sequence similarity to MCD peptide (see Fig. 1 ), is a potent neurotoxin when administered by intraventricular injection (1, 13) . At nanomolar concentration, apamin specifically inhibits a particular class of calcium-dependent potassium channels (14, 15) . Evidence has been presented that mammalian brain contains a peptide with biological activities similar to apamin (16) .


Figure 1: Amino acid sequences of apamin (A) and MCD peptide (B). Carboxyl-terminal amides are marked (NH). The CNCK-sequences found in both peptides are underlined. The arrow above the sequences indicates the length and orientation of the oligonucleotides used for the PCR and screening experiments.



Using cDNA cloning techniques, the structure of the precursors of melittin (17) , phospholipase A(4) , and hyaluronidase (6) have been elucidated. Here we present the sequence of the cloned cDNAs and of the gene encoding the precursors of MCD peptide and apamin. The sequence data suggest that the gene evolved from a common ancestor through a rather unusual partial duplication.


EXPERIMENTAL PROCEDURES

Isolation of mRNA from Venom Glands and cDNA Synthesis

Poly(A)-rich RNA was isolated from venom glands of worker bees as previously described (6) . First strand cDNA was synthesized with 2.5 µg of RNA, the primer-adaptor TGATTCAGGATCCTATCGA(T) and reverse transcriptase (Superscript, Life Technologies, Inc.) using a modified version of the RACE protocol (18) . The reaction was diluted 25-fold with TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0). This cDNA pool was then used for the polymerase chain reaction (PCR) experiments. For amplification of the 3`-ends of the cDNAs derived from the mRNAs encoding the precursors of apamin and MCD peptide, degenerate oligonucleotides encoding the last four amino acids plus the glycine residue required for amidation were used. These were Apa2 coding for Cys-Gln-Gln-His-Gly (TGT/C-CAA/G-CAA/G-CAT/C-GG, 14 mer) and MCD2 containing the codons for the peptide Cys-Gly-Lys-Asn-Gly (TGT/C-GGX-AAA/G-AAT/C-GG, 14 mer, X stands for all four bases). The conditions used for the two separate PCRs were the following. In a total volume of 50 µl, 10 µl of cDNA pool, 50 pmol of either Apa2 or MCD2, and 25 pmol of the adaptor Ada2 (TGATCAGGATCCTATCG, 17 mer), 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.01% gelatin, 1.5 mM magnesium chloride, 0.1% Triton X-100, 0.2 mM dNTPs (Pharmacia) and 0.2 unit of Hi-Taq polymerase (Vienna Laboratories) were mixed. After 30 cycles (40 s at 92 °C, 1 min at 50 °C (for MCD2), or 51 °C (for Apa2), and 1 min at 72 °C), an 150-bp fragment was amplified from the PCR in the presence of the Apa2 primer and a somewhat larger one from the other reaction. The fragments were eluted from the agarose gel, phosphorylated, blunt ended, and subcloned into the pBluescript vector (Stratagene). Both strands of the cloned cDNAs were sequenced by the chain termination method using the Sequenase 2.0 kit (U. S. Biochemical Corp.).

Preparation and Screening of a cDNA Library

The PCR fragments were labeled with [-P]dATP and [-P]dCTP using the Klenow polymerase and a random primed DNA labeling kit (Boehringer Mannheim). A cDNA library prepared from venom glands of worker bees (6) was screened with these fragments, and 24 positive clones were selected for further analysis.

Plasmid preparation and subsequent digestion with EcoRI and BamHI yielded insert sizes of 250-700 bp. Clones were further characterized by hybridization with the peptide-specific oligonucleotides Apa1 and MCD1, respectively. Apa1 coded for the amino-terminal apamin sequence Cys-Asn-Cys-Lys-Ala-Pro (TGT/C-AAT/C-TGT/C-AAA/G-GCX-CC, 17 mer), while MCD1 contained the codons of the fragment Lys-Cys-Asn-Cys-Lys-Arg (residues 2-7) (AAA/G-TGT/C-AAT/C-TGT/C-AAA/G-A/CG, 17 mer). Of the positive clones, about one-half hybridized with the Apa1 and the other with the MCD1 oligonucleotide. The clones with the largest inserts were sequenced using the chain termination method.

Isolation of Genomic Fragments

Genomic DNA was isolated as described by John et al.(19) . Bee pupae were homogenized in 10 ml of solution A (10 mM Tris-HCl, pH 7.6, 10 mM KCl, 10 mM magnesium chloride) containing 1.2% (v/v) Nonidet P-40. Nuclei were spun down and lysed in solution B (solution A containing 0.5 M NaCl and 0.5% SDS). After successive extraction with phenol (saturated with 1 M Tris-HCl, pH 8.0), phenol-chloroform-isoamyl alcohol (24:24:1), and chloroform-isoamyl alcohol (24:1), the DNA was precipitated with two volumes of ethanol. The washed DNA was then dissolved in TE buffer at a concentration of 0.2 µg/µl and stored at 4 °C.

Genomic DNA was amplified with two oligonucleotides: (a) AMG-5 (G-ATT-TCT/C-ATG-CTG/A-AGA-TG, 18 mer) containing the codons for the amino-terminal sequence (Me)t-Ile-Ser-Met-Leu-Arg-Cy(s) of both precursors; (b) AMG-3 (CCATTTTGATGAATCCAA, 18 mer, antisense) which bound to the 3`-untranslated region of both cDNAs. In a 50-µl reaction, 0.2-2 µg of genomic DNA and 25 pmol of each primer were used for the PCR (buffer as described above). After 30 cycles (40 s at 92 °C, 40 s at 52 °C, 2 min at 72 °C), two fragments containing about 400 bp (AMH1) and 1200 bp (AMH2) were specifically amplified. Both were subcloned into the pBluescript vector and sequenced.

Isolation of 5`-Upstream Regions

The promoter region preceding the first exon of the gene for the MCD peptide precursor was isolated by using inverse PCR technology (20) . Genomic DNA was digested with Sau3A, extracted with phenol-chloroform-isoamyl alcohol (24:24:1) and chloroform-isoamyl alcohol (24:1) and then precipitated. Self-ligation was performed under conditions favoring the formation of circles rather than concatamers, i.e. less than 1 µg of DNA/ml, incubation at 16 °C for 15 h. The reaction was stopped by extraction with phenol-chloroform-isoamyl alcohol (24:24:1) and chloroform-isoamyl alcohol (24:1), and DNA was precipitated with isopropyl alcohol and dissolved in TE buffer (25 µg/ml). The primers used for PCR were IP-1 (TACCATCGTCGGTGTTAC, nucleotides 79-62 of fragment AMH2, antisense) and IP-2 (AGACGTTGTCAACAGCAT, nucleotides 1128-1145 of AMH2, sense orientation). In a final volume of 50 µl, 2 µg of the religated DNA and 25 pmol each of IP-1 and IP-2 were incubated with Hi-Taq polymerase as described above. After 35 cycles (40 s at 92 °C, 40 s at 55 °C, 3 min at 72 °C), 10 µl of the reaction mixture were loaded onto an agarose gel, blotted, and hybridized with labeled AMG-3 primer. Labeling of AMG-3 was performed with T4 polynucleotide kinase in the presence of [-P]ATP. The hybridization conditions were as follows: 6 SSPE (1 SSPE = 150 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA) and 0.1% SDS at 30 °C for 90 min in the presence of about 10 cpm/ml of labeled AMG-3. Filters were washed three times with 6 SSPE and 0.1% SDS for 10 min at 35 °C. Under these conditions, a fragment containing about 1.4 kb hybridized with AMG-3. One-half of the PCR product was then loaded onto an agarose gel, and the barely visible band at 1.4 kb was eluted (gene clean II, BIO 101). This fragment was reamplified under the same conditions for 20 cycles, purified, blunt-ended with T4 DNA polmerase (BioLabs), and subcloned into the pBluescript vector. For sequence analysis, the Sequenase kit and a set of appropriate primers were used.

Sequence Analysis

For sequence comparisons, the Gene Works Program, version 2.3.1 (IntelliGenetics Inc.) was used on an Apple computer. Nucleotide sequences of cDNAs and genomic fragments have been deposited in the data base.


RESULTS

The Apamin Precursor

From poly(A)-rich RNA isolated from worker bee venom glands, cDNA was synthesized with the oligo(dT) adapter as primer for the reverse transcriptase reaction (23) . A degenerate oligonucleotide derived from the carboxyl-terminal pentapeptide of apamin (see Fig. 1A) was then used for the synthesis of the second strand. The nucleotide sequence of the 149-bp fragment thus obtained started at the 5`-end with the codons for the carboxyl end of apamin followed by a glycine and an in-frame stop codon (see Fig. 2A). This fragment was then labeled and used to screen a cDNA library prepared from worker bee venom glands. Two dozen positive clones were selected and rescreened with a degenerate oligonucleotide derived from the amino-terminal sequence Cys-Asn-Cys-Lys-Ala-Pro of apamin. About every other clone gave a positive signal in this second screening. Two of these clones, Apamin-3 and Apamin-6 containing inserts of 450 and 300 bp, respectively, were investigated further. The nucleotide sequence of the insert present in clone Apamin-6 is shown in Fig. 2A. Starting with the first ATG codon, this sequence contains a single open reading frame which encodes prepro-apamin comprising 46 amino acids. The cloned cDNA terminates at the 3`-end with a poly(A) segment which is preceded by the polyadenylation signal AATAAA. The following segments can be discerned in the precursor: (i) a signal sequence which probably terminates after serine 19; (ii) a proregion of 8 residues which ends in proline; (iii) the sequence of apamin; and (iv) a glycine residue required for formation of the carboxyl-terminal amide.


Figure 2: Nucleotide sequence of cloned cDNAs encoding the precursors of apamin (A) and MCD peptide (B). The deduced amino acid sequence of the precursors is shown in the single letter code above the nucleotide sequence. The predicted end of the signal peptides (29) and the end of the proregion are marked (), as well as the stop codons (///). The arrow () indicates the beginning of the segment where the sequences of both cDNAs are identical. The polyadenylation signals close to the 3`-ends are underlined.



The nucleotide sequence of the insert present in clone Apa3 was found to be largely identical to that of Apa6, except that an intron of 146 bp was present between the Met-Pro codons immediately preceding the sequence of apamin (see Fig. 2A). This intron is very rich in A + T (86%).

The Precursor for MCD Peptide

Using the RACE protocol (18) combined with the polymerase chain reaction, a fragment derived from the 3`-end of the mRNA for the precursor of MCD peptide was obtained. Surprisingly, the nucleotide sequence of this fragment was found to be identical to the 3`-end of the cloned cDNAs encoding the apamin precursor (Fig. 2B). In fact, most of the clones eliminated in the screen with the oligonucleotide specific for apamin (see above) were found to contain inserts encoding the precursor of MCD peptide. The inserts present in three of these clones were sequenced, and the results obtained with clone mcd-1 are shown in Fig. 2B. After the first initiation codon, this sequence had an open reading frame encoding a polypeptide of 50 amino acids. The precursor of MCD peptide contains a signal peptide of 19 amino acids, a propart of 8 resides terminating in serine, the sequence of mature MCD peptide and a carboxyl-terminal glycine from which the terminal amide is derived.

The cloned cDNAs for the precursors of apamin and MCD peptide show a high degree of homology at both ends. The 5`-untranslated region and the segment encoding the signal and the propart of the respective precursors are 85% identical. Even more striking is the fact that the 3`-ends of the two cloned cDNAs are completely identical. In view of these rather unusual sequence identities and similarities, it was considered of interest to investigate the structure of the corresponding genes.

Analysis of the Genes Encoding the Precursors of Apamin and MCD Peptide

Starting with genomic DNA, PCR experiments were performed to amplify parts of both genes simultaneously. Two oligonucleotides, AMG-5 and AMG-3, were synthesized that bind to regions close to the 5`- and the 3`-ends of both cDNAs, respectively (see ``Experimental Procedures''). Under suitable conditions, two fragments containing about 400 bp (AMH-1) and 1200 bp (AMH-2) were amplified. AMH-1 contains the three exons encoding the apamin precursor. Of the two introns, one was already known from the apamin-3 clone, while the second is located precisely in front of the common 3`-ends of the cDNAs. Sequence analysis of AMH-2 gave another surprising result. Starting from the 5`-end, this sequence comprises three exons of the precursor of the MCD peptide, interrupted by two short introns, then a larger intron of 400 bp, and finally the sequence of AMH-1. The genomic sequence encoding the two precursors is schematically shown in Fig. 3.


Figure 3: Schematic representation of the genes for the MCD peptide and apamin precursors. The structure of the genes was deduced from the sequence of AMH1 and AMH2. Exons are drawn as rectangles, introns as a thick line. The stop codons in two exons are marked by dots. Above and below the genomic sequence, the postulated splicing patterns yielding the mature mRNAs for the two precursors are indicated.



The gene for the precursor of the MCD peptide contains two introns at the same positions as the apamin precursor gene. All these introns are rich in A + T (84-88%). The second intron of both genes contains 81 bp, of which 51 (63%) are identical. The first introns of the two genes are, however, of different length, 127 bp for the apamin and 100 bp for the MCD peptide gene, respectively, and only limited sequence similarity close to the ends is discernible.

The postulated splicing scheme depicted in Fig. 3would yield the two mRNAs containing identical 3`-ends. In this scheme, two exons and two introns of the apamin gene form part of the third intron of the MCD gene.

The 5`-Upstream Region of the MCD Peptide Gene

The intron preceding the first exon of the apamin precursor contains a putative TATA box (see ``Discussion''). For the analysis of the corresponding region preceding the first exon of the gene for the MCD peptide precursor, we used the inverse PCR (25) . Genomic bee DNA was digested with Sau3A and religated under conditions favoring the formation of circles. Using the oligonucleotides IP-1 and IP-2 (see ``Experimental Procedures'') several fragments were amplified including the 1.4-kb fragment IPS-1.4. This fragment contained on the two sides of the GATC sequence recognized by the Sau3A restriction endonuclease about 330 bp derived from the 5`-side and 800 bp from the 3`-side of the genes for the two precursors. In Fig. 4, this 5`-sequence is compared with the sequence of the region that precedes the first exon of the apamin gene. The two sequences are 81% identical and both contain a TATA box 72 bp (MCD gene) or 76 bp (apamin gene) upstream from the ATG initiating codon. This provides further support for the assumption that the two genes originated from a partial duplication of a primordial gene.


Figure 4: Comparison of 5`-regions of the two genes. The genomic nucleotide sequences preceding the initiating methionine of the precursors for MCD peptide (upper lines) and apamin (lower lines) are shown. The latter sequence starts with the end of the third exon (underlined) of the MCD precursor gene (see Fig. 3), followed by the sequence of the following intron. The start of the cloned cDNAs is indicated by (<), and putative TATA boxes are marked by dots above the sequence. Identities are marked (filled squares); gaps (-) were introduced to maximize homology.




DISCUSSION

The precursors for apamin and MCD peptide are unusual for two reasons. First, they are extremely small, containing only 46 and 50 amino acids, respectively. It has previously been shown that precursor polypeptides comprised of only 60-80 amino acids, such as those for melittin from bee venom, cecropin from moth hemolymph, and PGLa from amphibian skin, can be transported across the membrane of the endoplasmic reticulum via a post-translational route which is independent of signal recognition particle and docking protein (21-23). Whether this is also true for the even smaller precursors of these bee venom peptides remains to be seen. The second aspect in which these precursors differ from the numerous other propeptides and proproteins that have been analyzed in recent years is the processing reactions which have to take place to obtain the mature peptide. The conversion of proapamin and pro-MCD peptide to the final products requires hydrolysis of Ser/Cys and Pro/Ile bonds, respectively. The endo- and/or exopeptidases catalyzing these reactions are not known. It has been shown that the liberation of melittin from its precursor proceeds via stepwise cleavage of dipeptides. A dipeptidyl aminopeptidase cleaving after proline and alanine residues participates in the processing of promelittin (24) as well as of several precursors from other sources (25) . However, it is unlikely that this exopeptidase could hydrolyze the proparts of the precursors of apamin or MCD peptide. In this context, it is noteworthy that liberation of hyaluronidase, another bee venom constituent, from its precursor requires hydrolysis of a Thr/Pro bond (6) , again a most unusual site for proteolysis. Generally, it appears likely that processing reactions in invertebrates are much more diverse than in vertebrate cells (see e.g. Ref. 26).

Apamin and MCD peptide have different biological activities, yet they show some sequence similarity (see Fig. 1). This homology is much more pronounced when one compares the sequence of the cloned cDNAs encoding these precursors. The 5`-untranslated region and the segments encoding the signal and the propeptides of the two precursor are 85% identical. What was, however, striking was the fact that the 3`-ends of the mRNAs for these precursors were found to be identical. This raised the intriguing possibility that a common 3`-exon was present in both mRNAs. Our analysis of the gene encoding these precursors has corroborated this notion.

The genomic organization was shown to be as follows. The first three exons of the precursor for the MCD peptide, two exons of the apamin precursor, and finally the 3`-exon were present in both mRNAs. For the apamin precursor, this last exon contains the genetic information for the COOH-terminal hexapeptide of the final product, whereas in the case of the MCD precursor, this exon is entirely within the 3`-untranslated region. The introns in the two genes occur in homologous positions: the first is located between the penultimate and the ultimate amino acid of the propart, while the second is inserted before the sixth residue from the carboxyl end. Moreover, these second introns are of identical length each containing 81 bp.

These results demonstrate that the 5`-region of the gene for the apamin precursor forms part of the third intron of the MCD peptide gene. This sequence is homologous to the upstream sequence preceding the latter gene. In fact a TATA box is present in both instances about 75 bp prior to the initiating ATG codons. We thus assume that the two genes have a transcription start site close to these TATA boxes. The primary transcript starting at the first TATA box would then be spliced in such a way that two exons and introns of the apamin gene are excised as part of the third intron of the MCD precursor gene. As shown schematically in Fig. 3, this would yield the mRNA encoding the precursor of the MCD peptide. Initiation of transcription at the the second TATA box and subsequent splicing of the two introns gives rise to the mRNA for the apamin precursor. This would also explain the fact that both mRNAs terminate in the same exon sequence. Apamin and MCD peptide are present in about equal amounts in the venom of worker bees, which indicates that both promoters are used with roughly the same frequency. They may also be controlled jointly, since only trace amounts of the two mRNAs can be detected on Northern blots with total RNA from queen bee venom glands.()

Both the origin of new genes by duplications and the generation of multiple products from one gene through alternative splicing have been documented in numerous instances. In addition, several cases of genes within other genes, particularly within introns of other genes, have been described. This was first shown for the Gart gene of Drosophila, which contains a gene for a pupal cuticle protein in one of its introns (27) . Last, a few examples are known where transcription initiation sites within a gene lead to the formation of smaller polypeptides. For example, a number of dystrophin-related proteins have been described which are encoded by mRNAs transcribed from the same gene by the use of an alternative internal promoter (28) . The common gene for the bee venom constituents MCD peptide and apamin apparently represents a new variation on the theme of gene organization with some similarity to these other cases. It could have originated from a primordial MCD precursor gene through, e.g. unequal crossing over, whereby the promoter region, two exons and two introns of this gene were duplicated. Transcription from both promoters then yields the two mRNAs encoding different components of honeybee venom.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Cellular Biochemistry and Biophysics Program, Sloan Kettering Institute, 1275 York Ave., Box 251 New York, NY 10021. Tel.: 212-639-8545; Fax: 212-717-3604.

The abbreviations used are: MCD, mast cell degranulating; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s).

M. Gmachl and G. Kreil, unpublished experiments.


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