Evidence for the major role of PH4⍺EFB in the prolyl 4-hydroxylation of Drosophila collagen IV

Ishikawa, Yoshihiro; Toups, Melissa A; Elkrewi, Marwan N; Zajac, Allison L.; Horne-Badovinac, Sally; Matsubayashi, Yutaka

Evidence for the major role of PH4⍺EFB in the prolyl 4-hydroxylation of Drosophila collagen IV

Ishikawa Y, Toups MA, Elkrewi MN, Zajac AL, Horne-Badovinac S, Matsubayashi Y. 2025. Evidence for the major role of PH4⍺EFB in the prolyl 4-hydroxylation of Drosophila collagen IV. Matrix Biology. 141, 101–113.

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https://doi.org/10.1016/j.matbio.2025.09.002 [Published Version]

DOI

10.1016/j.matbio.2025.09.002

Journal Article | Epub ahead of print | English

Scopus indexed

Author

Ishikawa, Yoshihiro; Toups, Melissa A^ISTA ; Elkrewi, Marwan N^ISTA ; Zajac, Allison L.; Horne-Badovinac, Sally; Matsubayashi, Yutaka

Department

Vicoso Group

Grant

The highjacking of meiosis for asexual reproduction

Abstract

Collagens are fundamental components of extracellular matrices, requiring precise intracellular post-translational modifications for proper function. Among the modifications, prolyl 4-hydroxylation is critical to stabilise the collagen triple helix. In humans, this reaction is mediated by collagen prolyl 4-hydroxylases (P4Hs). While humans possess three genes encoding these enzymes (P4H⍺s), Drosophila melanogaster harbour at least 26 candidates for collagen P4H⍺s despite its simple genome, and it is poorly understood which of them are actually working on collagen in the fly. In this study, we addressed this question by carrying out thorough bioinformatic and biochemical analyses. We demonstrate that among the 26 potential collagen P4H⍺s, PH4⍺EFB shares the highest homology with vertebrate collagen P4H⍺s. Furthermore, while collagen P4Hs and their substrates must exist in the same cells, our transcriptomic analyses at the tissue and single cell levels showed a global co-expression of PH4⍺EFB but not the other P4H⍺-related genes with the collagen IV genes. Moreover, expression of PH4⍺EFB during embryogenesis was found to precede that of collagen IV, presumably enabling efficient collagen modification by PH4⍺EFB. Finally, biochemical assays confirm that PH4⍺EFB binds collagen, supporting its direct role in collagen IV modification. Collectively, we identify PH4⍺EFB as the primary and potentially constitutive prolyl 4-hydroxylase responsible for collagen IV biosynthesis in Drosophila. Our findings highlight the remarkably simple nature of Drosophila collagen IV biosynthesis, which may serve as a blueprint for defining the minimal requirements for collagen engineering.

Publishing Year

2025

Date Published

2025-09-20

Journal Title

Matrix Biology

Publisher

Springer Nature

Acknowledgement

This project was supported by the All May See Foundation 7031,182 to YI, the Louisiana Board of Regents Support Fund: Research Competitiveness Subprogram to MAT, Austrian science fund (FWF) as part of the SFB Meiosis consortium FWF SFB F88-10 to Beatriz Vicoso (supported ME), American Heart Association 16POST2726018 and American Cancer Society 132,123-PF-18–025–01-CSM postdoctoral fellowships to ALZ, National Institutes of Health R01 GM136961 and R35 GM148485 to SH-B, and the Academy of Medical Sciences/the Wellcome Trust/ the Government Department of Business, Energy and Industrial Strategy/the British Heart Foundation/Diabetes UK Springboard Award SBF008\1115 to YM. Computational analyses of single-nucleus transcriptome data were performed on the high performance computer (HPC) at Bournemouth University, the HPC at Institute of Science and Technology Austria, and the high-performance computational resources provided by the Louisiana Optical Network Infrastructure (http://www.loni.org). The authors are grateful to the researchers who published the transcriptome datasets [48,49,52,55] that became the essential bases for this study, to FlyBase for curating the datasets in an easily accessible format, and the Drosophila Genomics Resource Center (DGRC), supported by NIH grant 2P40OD010949, for providing the D17 cell line used in this research. The authors thank Kristian Koski (University of Oulu, Finland) for crucial advice on the domain structure of collagen P4H⍺s, and Ryusuke Niwa and Ryo Hoshino (University of Tsukuba, Japan) for helpful discussions on SP.

Volume

141

Page

101-113

ISSN

0945-053X

eISSN

1569-1802

IST-REx-ID

20404

Cite this

Ishikawa Y, Toups MA, Elkrewi MN, Zajac AL, Horne-Badovinac S, Matsubayashi Y. Evidence for the major role of PH4⍺EFB in the prolyl 4-hydroxylation of Drosophila collagen IV. Matrix Biology. 2025;141:101-113. doi:10.1016/j.matbio.2025.09.002

Ishikawa, Y., Toups, M. A., Elkrewi, M. N., Zajac, A. L., Horne-Badovinac, S., & Matsubayashi, Y. (2025). Evidence for the major role of PH4⍺EFB in the prolyl 4-hydroxylation of Drosophila collagen IV. Matrix Biology. Springer Nature. https://doi.org/10.1016/j.matbio.2025.09.002

Ishikawa, Yoshihiro, Melissa A Toups, Marwan N Elkrewi, Allison L. Zajac, Sally Horne-Badovinac, and Yutaka Matsubayashi. “Evidence for the Major Role of PH4⍺EFB in the Prolyl 4-Hydroxylation of Drosophila Collagen IV.” Matrix Biology. Springer Nature, 2025. https://doi.org/10.1016/j.matbio.2025.09.002.

Y. Ishikawa, M. A. Toups, M. N. Elkrewi, A. L. Zajac, S. Horne-Badovinac, and Y. Matsubayashi, “Evidence for the major role of PH4⍺EFB in the prolyl 4-hydroxylation of Drosophila collagen IV,” Matrix Biology, vol. 141. Springer Nature, pp. 101–113, 2025.

Ishikawa, Yoshihiro, et al. “Evidence for the Major Role of PH4⍺EFB in the Prolyl 4-Hydroxylation of Drosophila Collagen IV.” Matrix Biology, vol. 141, Springer Nature, 2025, pp. 101–13, doi:10.1016/j.matbio.2025.09.002.

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