Abstract
Matrix proteins play important roles in shell formation. Our group firstly isolated three cDNAs encoding lysine-rich matrix protein from Pinctada fucata in 2006. However, the functions of KRMPs are not fully understood. In addition, KRMPs contain two functional domains, the basic domain and the Gly/Tyr domain respectively. Based on the modular organization, the roles of their two domains were poorly characterized. Furthermore, KRMPs were then reported in other two species, P. maxima and P. margaritifera, which indicated that KRMPs might be very important for shell formation. In this study, the characterization and function of KRMP-3 and its two functional domains were studied in vitro through purification of recombinant glutathione S-transferase tagged KRMP-3 and two KRMP-3 deletion mutants. Western blot and immunofluorescence revealed that native KRMP-3 existed in the EDTA-insoluble matrix of the prismatic layer and was located in the organic sheet and the prismatic sheath. Recombinant KRMP-3 (rKRMP-3) bound tightly to chitin and this binding capacity was duo to the Gly/Tyr-rich region. rKRMP-3 inhibited the precipitation of CaCO3, affected the crystal morphology of calcite and inhibited the growth of aragonite in vitro, which was almost entirely attributed to the lysine-rich region. The results present direct evidence of the roles of KRMP-3 in shell biomineralization. The functional rBR region was found to participate in the growth control of crystals and the rGYR region was responsible to bind to chitin.
Highlights
Mollusk shell formation is a typical biomineralization process, in which mineral crystals accumulate in a controlled manner [1,2,3]
Sequence-verified pGEX-4T-1 vectors containing either the KRMP-3 or basic region (BR) or Gly/Tyr-rich region (GYR) were used for expression in E. coli (Fig 1A)
The major bands of rKRMP3, rBR and rGYR on the gel migrated between 30 and 43 kDa, which correspond to the molecular masses of the fusion constructs: 35 kDa, 30.8 kDa and 30.2 kDa (26 kDa GST plus either 9, 4.8 or 4.2 kDa, respectively)
Summary
Mollusk shell formation is a typical biomineralization process, in which mineral crystals accumulate in a controlled manner [1,2,3]. The organic matrix makes up less than 5% of the shell and is thought to be responsible for biocrystal synthesis. Roles of KRMP-3 in Biomineralization proteins, polysaccharide and lipids, it is thought that proteins are the major macromolecules that control the essential phases, including crystal nucleation, crystal orientation and the determination of crystal polymorph and crystal morphology [6]. Nearly 50 matrix protein sequences have been identified [6]. Most of these sequences were obtained through molecular biology methods. The expression and purification of recombinant proteins in vitro is imperative. Among the matrix proteins for whose full-length DNA sequences are available, only a small fraction of these proteins could be expressed. Various protein expression systems were screened and different expression conditions were optimized, the difficulty of obtaining recombinant matrix proteins still remains
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