Abstract
BackgroundHundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains. ZP domain proteins perform highly diverse functions, ranging from structural to receptorial, and mutations in their genes are responsible for a number of severe human diseases. Recently, PLAC1, Oosp1-3, Papillote and CG16798 proteins were identified that share sequence homology with the N-terminal half of the ZP domain (ZP-N), but not with its C-terminal half (ZP-C). The functional significance of this partial conservation is unknown.ResultsBy exploiting a highly engineered bacterial strain, we expressed in soluble form the PLAC1-homology region of mammalian sperm receptor ZP3 as a fusion to maltose binding protein. Mass spectrometry showed that the 4 conserved Cys residues within the ZP-N moiety of the fusion protein adopt the same disulfide bond connectivity as in full-length native ZP3, indicating that it is correctly folded, and electron microscopy and biochemical analyses revealed that it assembles into filaments.ConclusionThese findings provide a function for PLAC1-like proteins and, by showing that ZP-N is a biologically active folding unit, prompt a re-evaluation of the architecture of the ZP domain and its polymers. Furthermore, they suggest that ZP-C might play a regulatory role in the assembly of ZP domain protein complexes.
Highlights
Hundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains
Identification of additional protein sequences containing only ZP-N To investigate whether other proteins exist that contain only the N-terminal half of the ZP domain, we generated a profile hidden Markov model (HMM) of ZP-N to scan genomic and non-redundant sequence databases
No proteins containing only ZP-C were found in a parallel search with a corresponding HMM profile
Summary
Hundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains. The N-terminal subdomain (ZP-N) is thought to contain conserved Cys 1 to 4, disulfide-bonded with invariant 1–4, 2–3 connectivity. On the other hand, conserved Cys 5 to 8, located within the C-terminal subdomain (ZP-C), apparently adopt two alternative connectivities in different ZP domain proteins [3,6,7,8,9,10]. In type I ZP domain proteins with 8 Cys within the ZP domain, such as ZP3, the ZP-C connectivity is 5–7, 6–8; in type II (page number not for citation purposes). Type I (ZP3-like) ZP domain proteins appear to polymerise into filaments only in the presence of type II (ZP1/ZP2-like) ZP domain proteins, whereas the latter can form homopolymers
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