Three‐dimensional structures of avian beta‐microseminoproteins: insight from the chicken egg‐specific beta‐microseminoprotein 3 paralog

Abstract

Beta-microseminoproteins (MSMBs) are small disulfide-rich proteins that are conserved among vertebrates. These proteins exhibit diverse biological activities and were mainly reported to play a role in male fertility, immunity, and embryogenesis. In this work, we focused on the chicken MSMB3 protein that was previously depicted as an egg antibacterial protein. We report that MSMB3 protein is exclusively expressed in the reproductive tissues of laying hens (in contrast to chicken MSMB1 and MSMB2 paralogs), to be incorporated in the egg white during the process of egg formation. We also showed that chicken MSMB3 possesses highly conserved orthologs in bird species, including Neognathae and Palaeognathae. Chicken MSMB3 was purified from egg white using heparin affinity chromatography and was analyzed by top-down and bottom-up proteomics. Several proteoforms could be characterized, and a homodimer was further evidenced by NMR spectroscopy. The X-ray structure of chicken MSMB3 was solved for the first time, revealing that this protein adopts a novel dimeric arrangement. The highly cationic MSMB3 protein exhibits a distinct electrostatic distribution compared with chicken MSMB1 and MSMB2 structural models, and with published mammalian MSMB structures. The specific incorporation of MSMB3 paralog in the egg, and its phylogenetic conservation in birds together with its peculiar homodimer arrangement and physicochemical properties, suggests that the MSMB3 protein has evolved to play a critical role during the embryonic development of avian species. These new data are likely to stimulate research to elucidate the structure/function relationships of MSMB paralogs and orthologs in the animal kingdom.Beta-microseminoproteins (MSMBs) are nonglycosylated disulfide-rich miniproteins (molecular mass about 10 kDa) that have been identified in many animal species [1,2]. MSMB genes have been reported to exhibit strong conservation among vertebrates, although they share a relatively low level of sequence identity Abbreviations MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MSMB, microseminoprotein; nanoLC-MS/MS, nanoflow liquid chromatography-tandem mass spectrometry; PDB, protein data bank; PSP94, prostate secretory protein 94; SSP-2, small serum protein 2; TOCSY, total correlation spectroscopy.

Antimicrobial Proteins and Peptides in Avian Eggshell: Structural Diversity and Potential Roles in Biomineralization

“…MSMB genes are widely distributed among vertebrates ( 188 ). Three chicken paralogues (MSMB1, MSMB2, MSMB3), whose genes are localized on chromosome 6, have been described ( 189 ). Orthologs of these three genes are found in several bird species and, interestingly, the degree of conservation between avian MSMB3 orthologs is higher than that observed between avian MSMB1 or MSMB2 orthologs ( 188 ).…”

“…Of note, murine MSMB protein is more similar to avian MSMB2 than MSMB1 and MSMB3 ( 188 ). Given the apparent oviduct-specific expression of chicken MSMB3 ( 189 ), it is thought that the biological function(s) of avian MSMB3 might have functionally diverged from that of other vertebrate or mammalian MSMBs, towards a specific role in reproduction. The phylogeny and evolutionary description of MSMB gene(s) within reptiles remains to be investigated.…”

“…The X-ray crystal structure of MSMB3 ( 189 ) shows that it is composed of a N-terminal domain linked to a C-terminal domain by a linker peptide ( Figure 10A , left panel). The N-terminal domain has a four-stranded antiparallel β-sheet arranged in a Greek key motif whereas the C-terminal domain has one antiparallel β-sheet composed of two strands.…”

“…Similar to its human and porcine counterparts, chicken MSMB3 can be found as a homodimer in solution ( Figure 10B ). However, unlike human and porcine MSMB3 dimers in which each monomer associates in an edge-to-edge mode, MSMB3 monomers associate in a different pattern, forming most interactions between their respective C-terminal domains ( Figure 10B ) ( 189 ). It is not yet known whether the biologically active species is the monomer, the dimer, or both forms.…”

“…The physiological function of these proteins are not known, but MSMB3 purified from egg white displays antibacterial activities against L. monocytogenes and S. Enteritidis ( 38 ). It is noteworthy that chicken MSMB1 and MSMB2 remain uncharacterized because they are not yet available as purified proteins ( 189 ). Similar to OVAX, MSMB3 antibacterial activity is blocked by heparin, which suggests that the MSMB3 heparin-binding site is important for the interaction with bacteria.…”