In vivo expression of peptidylarginine deiminase in Drosophila melanogaster

Abstract

Peptidylarginine deiminase (PAD) modifies peptidylarginine and converts it to peptidylcitrulline in the presence of elevated calcium. Protein modification can lead to severe changes in protein structure and function, and aberrant PAD activity is linked to human pathologies. While PAD homologs have been discovered in vertebrates-as well as in protozoa, fungi, and bacteria-none have been identified in Drosophila melanogaster, a simple and widely used animal model for human diseases. Here, we describe the development of a human PAD overexpression model in Drosophila. We established fly lines harboring human PAD2 or PAD4 transgenes for ectopic expression under control of the GAL4/UAS system. We show that ubiquitous or nervous system expression of PAD2 or PAD4 have minimal impact on fly lifespan, fecundity, and the response to acute heat stress. Although we did not detect citrullinated proteins in fly homogenates, fly-expressed PAD4-but not PAD2-was active in vitro upon Ca2+ supplementation. The transgenic fly lines may be valuable in future efforts to develop animal models of PAD-related disorders and for investigating the biochemistry and regulation of PAD function.

Fig 1. Human PAD2 and PAD4 expression in E. coli and in vitro citrullination activity.

(A) Western blot for PAD2 (left) or PAD4 (right). E. coli were transformed with a PAD cDNA vector (+) or an empty vector control (-). Arrowheads point to bands specific to PAD-transformed E. coli. PAD4 was expressed as a GST-tagged fusion protein; the GST tag could be removed by addition of the appropriate protease. Non-specific labeling is also observed with both primary antibodies, based on the consistent bands seen in all lanes including the non-transformed controls. 10 μg of total protein was loaded per lane. (B) Citrullination activity of protein extracts from E. coli expressing PAD2 or PAD4, assessed in the presence (+) or absence (-) of 10 mM Ca²⁺. Citrullinated proteins were detected by Western blot.

Fig 2. PAD overexpression does not alter lifespan in D. melanogaster.

Ubiquitous (da-GAL4) or pan-neuronal (elav-GAL4) expression of (A) PAD2 or (B) PAD4 in female flies did not significantly affect lifespan. Similarly, overexpression of (C) PAD2 or (D) PAD4 did not consistently alter male survival. Control lines harbor only the GAL4 or the UAS-controlled PAD transgene. Two independent transgenic lines were assessed for each PAD (PAD2: PAD2_2 and PAD2_3; PAD4: PAD4_7 and PAD4_8). The log-rank p value is only shown when pairwise multiple comparisons reveal that survival curves differ between the experimental line and both controls.

Fig 3. PAD overexpression does not alter fecundity.

In reciprocal crosses of experimental flies with Canton-S (CS), fecundity was unaffected, as assessed by (A) the number of F1 progeny developed to adulthood and (B) the fraction of males in the F1 progeny.

Fig 4. PAD overexpression does not affect the response to heat stress.

(A) Average time to locomotor failure and (B) average time to recovery from acute hyperthermia was assessed in males (5–9 days old) individually housed in the Drosophila Activity Monitor.

Fig 5. Human PAD2 and PAD4 expression and citrullination activity from Drosophila larvae.

(A) Western blot for PAD2 (left) or PAD4 (right) using Drosophila larval protein extracts. Control lines harbor only the GAL4 or the UAS-controlled PAD transgene, while the experimental groups show PAD expression under the control of a ubiquitous GAL4 driver (da-GAL4). Arrowheads point to bands specific to PAD-expressing flies. Non-specific labeling is also observed with both primary antibodies, based on the consistent bands seen in all lanes including the negative controls. 30 μg of total protein was loaded per lane. (B) Detection of citrullinated proteins by Western blot of protein extracts from Drosophila larvae expressing PAD2 (left) or PAD4 (right). Cl-amidine (Cl-A, 1 mM) supplementation inhibits Ca²⁺-mediated PAD4 activity.

Fig 6. Human PAD2 expression and citrullination activity from Drosophila adults.

(A) Western blot for PAD2 using fly protein extracts. Control lines harbor only the GAL4 or the UAS-controlled PAD transgene, while the experimental group shows PAD2 expression under the control of a ubiquitous GAL4 driver (da-GAL4). Arrowhead points to a band specific to PAD2-expressing flies. Non-specific labeling is also observed, based on the consistent bands seen in all lanes including the negative controls. 25 μg of total protein was loaded per lane. (B) Detection of citrullinated proteins by Western blot of protein extracts from Drosophila adults expressing PAD2. As a positive control, citrullination activity of a protein extract from PAD2-expressing E. coli is also shown. Cl-amidine (Cl-A, 1 mM) supplementation inhibits E. coli-expressed PAD2 activity. (C) Detection of citrullinated proteins by Western blot of protein extracts from flies ubiquitously expressing PAD2, supplemented with or without a protein extract from E. coli. As a positive control, citrullination activity of a protein extract from PAD2-expressing E. coli is also shown. (D) Detection of citrullinated proteins by Western blot of protein extracts from E. coli expressing PAD2 mixed with or without control fly homogenate (not expressing PAD2). (E) Detection of citrullinated proteins by Western blot of protein extracts from control flies (not expressing PAD2) with or without purified recombinant PAD2 (rPAD2).