Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix

Abstract

Background: The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously.

Results: Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components.

Conclusions: The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins.

Figure 1

Purified S. purpuratus spicules visualized by scanning electron microscopy. A, section showing fragments of spicules prepared from pluteus larvae. Fragmentation is due to fractures caused by homogenization during purification. Maximal length of spicule fragments is 70-80 μm. B, higher magnification showing the clean surfaces of spicule fragments. C, cross-section of a fractured spicule with a diameter of 4 μm showing the concentric layers of mineral. D, deeper etching and higher magnification reveals presumed matrix fibers coursing through the mineral layers.

Figure 2

PAGE separation of spicule matrix proteins. The molecular weight of marker proteins is shown in kDa to the left. Sections excised for in-gel digestion are indicated to the right.

Figure 3

MS/MS spectrum of a SM30-D peptide. This spectrum shows the MS/MS analysis of a triply charged peptide confirming the annotated predicted sequence contained in SPU:000828. As to be expected almost all of the major y-ions correspond to preferential cleavages N-terminal to prolines. The N-terminal Gln is cyclized to pyroglutamine. The mass error was 0.11 ppm, the PEP score was 3.01E-46 and the Mascot score was 63. Spectra of other unique peptides are shown in additional file 6.