Seaver RW and Livingston BT (2015), Examination of the skeletal proteome of the bri...

ECB-ART-43848

Proteome Sci 2015 Feb 07;13:7. doi: 10.1186/s12953-015-0064-7.

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Examination of the skeletal proteome of the brittle star Ophiocoma wendtii reveals overall conservation of proteins but variation in spicule matrix proteins.

Seaver RW , Livingston BT .

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BACKGROUND: While formation of mineralized tissue is characteristic of many animal taxa, the proteins that interact with mineral are diverse and appear in many cases to be of independent origin. Extracellular matrix proteins involved in mineralization do share some common features. They tend to be disordered, secreted proteins with repetitive, low complexity. The genes encoding these proteins are often duplicated and undergo concerted evolution, further diversifying the repetitive domains. This makes it difficult to identify mineralization genes and the proteins they encode using bioinformatics techniques. Here we describe the use of proteomics to identify mineralization genes in an ophiuroid echinoderm, Ophiocoma wendtii (O. wendtii). RESULTS: We have isolated the occluded proteins within the mineralized tissue of the brittle star Ophiocoma wendtii. The proteins were analyzed both unfractionated and separated on SDS-PAGE gels. Each slice was analyzed using mass spectroscopy and the amino acid sequence of the most prevalent peptides was obtained. This was compared to both an embryonic transcriptome from the gastrula stage when skeleton is being formed and a tube foot (an adult mineralized tissue) transcriptome. Thirty eight proteins were identified which matched known proteins or protein domains in the NCBI databases. These include C-type lectins, ECM proteins, Kazal-type protease inhibitors, matrix metalloproteases as well as more common cellular proteins. Many of these are similar to those found in the sea urchin Strongylocentrotus purpuratus (S. purpuratus) skeleton. We did not, however, identify clear homologs to the sea urchin spicule matrix proteins, and the number of C-type lectin containing genes was much reduced compared to sea urchins. Also notably absent was MSP-130. CONCLUSIONS: Our results show an overall conservation of the types of proteins found in the mineralized tissues of two divergent groups of echinoderms, as well as in mineralized tissues in general. However, the extensive gene duplication and concerted evolution seen in the spicule matrix proteins found in the sea urchin skeleton was not observed in the brittle star.

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Genes referenced: LOC100887844 LOC594261 LOC752081 LOC756768

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	Figure 1. SDS-PAGE gel of O. wendtii proteins isolated from the adult skeleton. 1: MW markers, 2: O. wendtiiprotein. A, B and C indicate major protein bands excised for LC/MS/MS analysis.
	Figure 2. Distribution of identified proteins among selected types of proteins.
	Figure 3. Identification of a 24 kd skeletal protein with a C-type lectin domain. A. Example spectra identifying Contig 266 as encoding a protein occluded in the O. wendtii skeleton. The predicted domain structure is shown in B. C: The open reading frame of Contig 266 with amino acid sequences identified using LC/MS/MS in bold. The amino acids identified in the spectrum in A are underlined.
	Figure 4. Identification of a 21 kd protein with a C-type lectin domain. A. Example spectra identifying Contig 1464 as encoding a protein occluded in the O. wendtii skeleton. The predicted domain structure is shown in B. C: The open reading frame of Contig 1464 with amino acid sequences identified using MS/MS in bold. The amino acids identified in the spectrum in A are underlined.
	Figure 5. A Maximum Likelihood tree showing the relationships among O. wendtii and S. purpuratus spicule matrix proteins found in the skeletal proteomes or identified through BLAST searches of the S. purpuratus genome using O. wendtii proteins as query. Alignments are shown in Additional file 6 with Gblocks identified. O. wendtii proteins are in bold. Boxed proteins are those not found in the proteome; all others are found in skeletal proteomes. Proteins with *are those that had expanded repeats removed prior to alignment. 1 indicates two splice variants of the same gene. Bootstrap values of 50 or greater are showhn in red. Nodes with bootstrap values below 25 were collapsed. The accession numbers for the sequences used are as follows: [Genbank: XP_796721.1, NP_999775.1, NP_999630.1, NP_999804.1, XP_003726194.1, NP_999803.1, NP_999776.1, XP_781636.1, KJ999723, KJ999724, KJ999725, KJ999726, KJ999727].
	Figure 6. Identification of a protein with a novel domain structure. A. Example spectra identifying Contig 358 as encoding a protein occluded in the O. wendtii skeleton. The predicted domain structure is shown in B. C: The open reading frame of Contig 358 with amino acid sequences identified using MS/MS in bold. The amino acids identified in the spectrum in A are underlined.

References [+] :

Aiken, Unraveling metalloproteinase function in skeletal biology and disease using genetically altered mice. 2010, Pubmed