Neprilysins (neutral endopeptidases; Neps) belong to the M13 family of Zinc (II) dependent metallopeptidases and were first discovered in mammals (Turner et al., 2001). Typical mammalian Neps, including human Neprilysin (HsNep) encoded by the MME (Membrane Metalloendopeptidase) gene, have a short cytoplasmic N-terminal domain, a shorter hydrophobic transmembrane domain, and a long extracellular C-terminal domain that houses highly conserved motifs. Two of these motifs, HEXXH and EXXAD, are responsible for Zn (II) binding and the subsequent proteolytic cleavage of substrate peptides, typically occurring at the N-terminal side of a hydrophobic residue with a strong preference for Phe or Leu toward the C-terminus (Nalivaeva & Turner, 2013; Sitnik et al., 2014; Turner et al., 2000).

Typical mammalian Neps are normally expressed in a variety of cell types, including brush border cells of the kidneys, brain, neutrophils, lungs, synapses, along with adipocytes and smooth muscle cells present throughout gut tissue, to name a few. Neprilysin cleaves an array of peptides and controls cellular signals that impact the nervous, cardiovascular, inflammatory, immune systems, and nutrient homeostasis (Schling & Schäfer, 2002; Turner et al., 2001).

Phylogenetic studies have identified the presence of Nep orthologs in invertebrate organisms, including Drosophila melanogaster (Bland et al., 2008; Sitnik et al., 2014). Drosophila melanogaster contains 28 identified neprilysin and neprilysin-like genes (Meyer et al., 2021). Among them, Neprilysin-like 15 (Nepl15), a predicted secreted, catalytically inactive Nep, regulates glycogen and lipid storage in a sex-dependent manner. Previous work with Nepl15 ^ko (knock-out) flies revealed that glycogen and triacylglyceride storage were significantly reduced in adult male mutant flies, while significantly more glycogen storage was observed in adult female mutants, albeit the mutants consume the same amount of food as the controls (Banerjee et al., 2021). Interestingly, a previous study (Banerjee et al., 2021) and transcriptomics data from FlyAtlas2 (Leader et al., 2018) showed that the Nepl15 transcript is differentially expressed in both larval and adult fly organs, with higher expression in larval fat body and adult head, heart, and fat body. Overall, FlyAtlas shows that the whole adult male body expresses 4.2 times more Nepl15 than the whole adult female body. Additionally, overexpression of Nepl15 in the fat body increased triglyceride levels only, whereas its overexpression in the gut increased glycogen levels only in adult male flies, suggesting Nepl15 has tissue-specific variable effects (Banerjee et al., 2021). Interestingly, the fly database (FlyBase.org) reports that the Nepl15 gene in D. melanogaster ISO1 MT (dm6) ( y ¹ ; cn ¹ bw ¹ sp ¹ ) strain has no intron and produces only one transcript. However, 10 other fly Neprilysin and Neprilysin-like genes encode more than one transcript (Table 1) (FlyBase.org).

Therefore, we asked the following questions: Are there any unreported Nepl15 transcript variants present in wild-type (Oregon-R) adult male and female flies that produce different Nepl15 protein isoforms in the two sexes? Are there any differences in the 5′ and 3′ untranslated regions (UTRs) in the Nepl15 transcript which may contribute to its post-transcriptional regulation? To answer these questions, we isolated total RNA from the whole-body of adult male and virgin female Oregon-R flies (FBsn0000276) and synthesized cDNA. Next, we took PCR based approach to amplify the Nepl15 CDS (coding sequence, 2061 bps) and its transcript (2213 bps including the 5′ and 3′ UTRs), followed by sequencing them and aligning the sequences with the corresponding sequences in FlyBase.

We did not find any nucleotide sequence difference at the 5′ and 3′ UTRs of the Oregon-R and FlyBase Nepl15 transcript sequences. However, sequencing results show that the Oregon-R Nepl15 CDS from male and female flies contains the exact 7-nucleotide sequence changes compared to the FlyBase sequence. These changes translate into only 5 amino acid changes, among which 3 changes include amino acids with different properties (Table 2) ( Figure 1A ). The Oregon-R adult male and female Nepl15 (ORNepl15M and ORNepl15F) protein sequences were aligned with the FlyBase Neprilysin1 (FBNep1) and Nepl15 (FBNepl15) protein sequences using ClustalW to show the changed 5 amino acid residues (Table 2) ( Figure 1A ). Further bioinformatics analysis of the Oregon-R Nepl15 protein reveals that the amino acid changes did not impact the Nepl15 predicted cleavage site (between amino acids 22 and 23) (SignalIP and DeepTMHMM) (Hallgren et al., 2022; Teufel et al., 2022), and extracellular localization (DeepLoc) (Ødum et al., 2024). Additional analysis of 3D models of the FlyBase Nepl15 ( Figure 1B, blue ribbon diagram) and Oregon-R Nepl15 proteins utilizing AlphaFold 3 (Abramson et al., 2024) shows no significant change in their structures in their overlays (blue and purple ribbons depict the Nepl15 3D protein models in FlyBase strain and Oregon-R, respectively) (Figures 1 C ₁ – C ₃ ).

Collectively, these results reveal that a slightly different amino acid sequence in the Nepl15 protein is present in our lab-grown wild-type Oregon-R flies as compared to the FlyBase Nepl15 protein, indicative of a strain-specific amino acid sequence variation in the same protein. Bioinformatic analyses do not indicate any major structural or functional shifts in the Oregon-R Nepl15 protein due to these changes. Thus, our study confirms the presence of only one Nepl15 transcript version in D. melanogaster Oregon-R adult male and female flies. Although, the changes in the nucleotide sequences within the Oregon-R Nepl15 codons and corresponding amino acid changes in the Nepl15 protein may not impact the protein's localization, structure, and function, still our research outcome can contribute to future investigations in two significant ways: (i) this report will help to perform primer design, PCR, qPCR, gene cloning, guide DNA design for CRISPR, and site specific mutation accurately for Nepl15 gene in Oregon-R fly strain; and (ii) if researchers use a Drosophila species or strain other than the one used in a database or previously published work, our result recommends that it is important to verify the DNA sequence before designing the components of molecular biology based experiments.

Drosophila melanogaster husbandry

Drosophila melanogaster Oregon-R strain was maintained on Bloomington's fly food formulation (Genesee Scientific Nutri-Fly_66-116) at 25 °C. 5 to 7 days old male and virgin female adult flies were used for RNA extraction.

FlyBase Data:

The FlyBase website, http://flybase.org, was used to retrieve the Nepl15 transcript and CDS sequences. The genome release number is FB2026_01 (March 12, 2026).

RNA isolation and cDNA synthesis

Total RNA was extracted from 5 to 7 days old, 25 male and 25 virgin female Oregon-R adult flies using TRI reagent (Sigma T9424) (Banerjee et al., 2021). The RNA sample purity was validated by measuring 260/280 and 230/280 ratios, and the RNA integrity was tested by gel electrophoresis. The cDNA synthesis was then performed using 5x iScript Reaction Mix (Bio-Rad 1708889) following the manufacturer's protocol.

PCR and Purification

Specific forward (FP) and reverse primer (RP) pairs (Table 3) were designed using the Serial Cloner (v2.6.1) (Branco & Choupina, 2021) to amplify Nepl15 CDS and transcript using the OR male and female cDNA templates separately. The primer sequences were designed based on the FlyBase Nepl15 CDS and Transcript sequences. PCR was performed using Q5 High-Fidelity 2X Master Mix (New England Biolabs M0492S) following the manufacturer's protocol. The PCR products were purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel 740609) following the manufacturer's instructions.

Table 3. List of primers used for PCR.

DNA Sequencing Analysis

We designed several primers (listed in Table 4) to cover the entire length of the Nepl15 CDS or transcript. Sanger sequencing of the purified PCR samples was performed by Azenta Life Sciences (genewiz.com). The high-quality sequence contigs were aligned to the corresponding FlyBase sequence using SerialCloner (v2.6.1) (Branco & Choupina, 2021).

Table 4. List of primers used for sequencing.

Protein sequence analysis and other bioinformatics analysis

Changes in the Oregon-R Nepl15 CDS sequences compared to the FlyBase sequence were identified using the Serial Cloner (v2.6.1) (Branco & Choupina, 2021). Variations in the Nepl15 codon sequences between Oregon-R CDS and FlyBase CDS sequences were analyzed using Serial Cloner's built-in BLASTn and BLASTp alignment tools. Next, Clustal Omega (Madeira et al., 2024) was used to align the FlyBase Neprilysin 1 (DmNep1, a typical fly neprilysin, orthologous to human neprilysin), Oregon-R male and female Nepl15, and FlyBase Nepl15 protein sequences to identify changes in Oregon-R Nepl15 protein sequences. The newly sequenced Oregon-R Nepl15 protein sequence was analyzed for any change in its subcellular localization, signal sequence site, and 3D structure compared to those of the FlyBase-reported Nepl15 protein sequence using different bioinformatics tools like DeepLoc2.1 (Ødum et al., 2024), SignalP – 6.0 (Teufel et al., 2022), and DeepTMHMM – 1.0 (Hallgren et al., 2022), respectively.

AlphaFold 3 Protein Prediction

Protein structure predictions for Nepl15 were generated using AlphaFold 3 within the ChimeraX interface. The amino acid sequence of the Oregon-R Nepl15 protein was used as input. Structure prediction was performed in ChimeraX (version 1.11.1) using the following workflow: Tools to Structure Prediction to AlphaFold, after which the protein sequence was entered into the “Sequence” field and submitted for prediction. The “don't minimize” parameter was selected before the sequence in the Google Collab window to prevent minimization during model generation, consistent with previously described protocols. Multiple models were generated, and model confidence was evaluated using predicted aligned error (PAE) metrics. The structure with the highest overall confidence, defined by the lowest PAE values and highest predicted local distance difference test scores, was automatically selected for further visualization and structural alignment analyses (Abramson et al., 2024; Meng et al., 2023).

All reagents' names, with the company names and catalog numbers, are mentioned in the methods.