Molecular adaptations in MMP genes support lung elasticity and diving adaptations in cetaceans

Abstract

Cetaceans are a unique group of marine mammals that have evolved from terrestrial to fully aquatic life. During diving, they experience extreme physiological challenges, including lung collapse, limited gas exchange, and the risk of decompression-related injuries. The matrix metalloproteinase (MMP) gene family plays a central role in extracellular matrix (ECM) remodeling, vascular repair, and inflammatory responses, and is also involved in the formation and maintenance of elastic fibers-key components that contribute to lung elasticity. Enhanced lung elasticity is thought to facilitate reversible lung collapse and efficient blood shift during dives, ultimately reducing nitrogen uptake and the potential risk of decompression sickness (DCS). In this study, we analyzed 1,058 genes from 46 species, focusing on cetaceans and other diving marine mammals, with terrestrial mammals as a reference group. Our results reveal that the MMP gene family has undergone positive selection in cetaceans, with nine genes exhibiting accelerated evolution. Notably, we identified a cetacean-specific N319S mutation in the Fibronectin type-II domain of MMP9, which impairs collagen-binding and degradation, as confirmed by Western blot analysis. Mass spectrometry further revealed an increased number of post-translational modifications in cetacean MMP9 compared to terrestrial mammals, with several modifications overlapping the mutation sites. These findings suggest that adaptive changes in MMPs may enhance elastic fiber dynamics and vascular remodeling in cetaceans, contributing to physiological adaptations such as improved lung compliance and resilience to diving-related stress, including reduced susceptibility to DCS.

Keywords: Convergent evolution; Diving adaptations; Marine mammals; Pulmonary fibrosis.

Fig. 1

Positive selection and accelerated evolution of MMP genes in marine mammals and their ancestral lineages. (A) Genes undergoing accelerated evolution in marine mammals within the MMP gene family. (B) Genes under positive selection within the MMP gene family in both marine mammals and their ancestral lineages. In the phylogenetic tree, blue represents marine mammals, and orange represents terrestrial mammals. circles and pentagons represent the free-ratio and branch-site models, respectively. (C) GO enrichment analysis of genes with accelerated evolution. (D) KEGG enrichment analysis of genes with accelerated evolution. MMP genes under accelerated evolution are mainly enriched in processes related to extracellular matrix synthesis and degradation, as well as collagen breakdown and formation

Fig. 2

Convergent sites in marine mammals and cetaceans-specific amino acid mutations. (A) Convergent amino acid substitution of MMP24 in marine mammals; (B) Cetacean-specific amino acid mutation sites in MMPs and their corresponding important functional domains

Fig. 3

Regression analysis of gene evolution rates with maximum diving depth and time. (A) Regression of maximum diving depth against the evolution rate of MMP16 (R²=0.2408, P = 0.01186); (B) Regression of maximum diving time against the evolution rate of MMP16 (R²=0.3057, P = 0.00448); (C) Regression of maximum diving depth against the evolution rate of MMP9 (R² = 0.3381, P = 0.00268). (D) Regression of maximum diving depth against the evolution rate of MMP25 (R² = 0.2026, P = 0.02045); (E) Regression of maximum diving depth against the evolution rate of MMP17 (R² = 0.3316, P = 0.00298);(F) Regression of maximum diving depth against the evolution rate of MMP11 (R² = 0.3120, P = 0.00406)

Fig. 4

MMP9 overexpression, protein modifications, and collagen degradation. (A) The highest XCorr protein spectrum peak identified from the overexpression of T. truncatus MMP9. (B) In cetaceans, the specific amino acid sites of MMP9 undergo post-translational modifications: the 319 S site is modified with HexNAc, the 532 S site is modified with Phospho and HexNAc, the 661 M site is modified with Oxidation, and the 568 S site is modified with Phospho and HexNAc. (C) The highest XCorr protein spectrum peak identified from the overexpression of B. taurus MMP9. (D) Collagen I degradation ability of T. truncatus and B. taurus MMP9 overexpressed in A549 cells. The blots/gels displayed in the figure represent cropped versions. Full-length blots/gels are presented in Fig S4

Fig. 5

Migration ability of A549 cells after MMP9 overexpression. (A) Wound healing status after 0–24 h of overexpression of T. truncatus and B. taurus genes. (B) Quantitative analysis of wound healing rate, ns: Not significant; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ***: P < 0.0001. P value of 12 h (Tur&Bos; Tur&NC; Bos&NC: 0.0059; 0.0022; 0.0098); P value of 24 h (Tur&Bos; Tur&NC; Bos&NC: 0.0241; 0.001; 0.001)