Limits...
Proteome analysis of shell matrix proteins in the brachiopod Laqueus rubellus.

Isowa Y, Sarashina I, Oshima K, Kito K, Hattori M, Endo K - Proteome Sci (2015)

Bottom Line: We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins.The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences.Our study suggests an independent origin and unique mechanisms for brachiopod shell formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan.

ABSTRACT

Background: The calcitic brachipod shells contain proteins that play pivotal roles in shell formation and are important in understanding the evolution of biomineralization. Here, we performed a large-scale exploration of shell matrix proteins in the brachiopod Laqueus rubellus.

Results: A total of 40 proteins from the shell were identified. Apart from five proteins, i.e., ICP-1, MSP130, a cysteine protease, a superoxide dismutase, and actin, all other proteins identified had no homologues in public databases. Among these unknown proteins, one shell matrix protein was identified with a domain architecture that includes a NAD(P) binding domain, an ABC-type transport system, a transmembrane region, and an aspartic acid rich region, which has not been detected in other biominerals. We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins. The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences.

Conclusions: Our study suggests an independent origin and unique mechanisms for brachiopod shell formation.

No MeSH data available.


Abundance index of shell matrix proteins identified in this study
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4536745&req=5

Fig5: Abundance index of shell matrix proteins identified in this study

Mentions: SDS-PAGE of the soluble organic matrix extracted from the secondary layer showed two major bands (< 6.5 and 12 kDa) and a minor band of 62 kDa when stained with CBB (Fig. 1a). Silver staining showed a major band at 35 kDa and many minor bands in addition to the three bands stained with CBB (Fig. 1b). Transcriptome sequencing in mantle tissue generated a total of 125,437 reads. To identify shell matrix proteins from Laqueus rubellus, we performed a proteome analysis of the material from these different sources, namely (1) the soluble organic matrix from the whole shell, (2) insoluble organic matrix from the whole shell and (3) soluble organic matrix from the shell secondary layer. The generated MS/MS data were subjected to SEQUEST searches against the protein sequence database obtained from the transcriptome analysis. As a result, we identified 40 shell matrix proteins (Tables 1, 2, 3, 4 and Fig. 2). Among these proteins, 18 proteins were identified from the soluble organic matrix of the shell secondary layer, and the soluble and insoluble organic matrix from the whole shell (Table 1). While five proteins were identified from both the soluble and insoluble organic matrix of the whole shell, 17 proteins were only identified from either the soluble or insoluble organic matrices of the whole shell (Tables 2, 3, 4). A total of 22 proteins were deduced to be possibly complete sequences, because these sequences have a stop codon and either an in-frame start codon just after an in-frame stop codon in the 5′ region of the sequence or a potential signal peptide (Tables 1, 2, 3, 4). A blast search against the GenBank non-redundant database showed that 36 out of the 40 shell matrix proteins have no annotated homologous sequences. Among the proteins that have sequence homology, isotig 00281 showed relatively high sequence similarity with MSP130, which is a skeletal protein identified from sea urchins (Fig. 3) [20, 21]. Isotig 01587, isotig 00949, and isotig 00959 showed high sequence similarity with extracellular copper/zinc superoxide dismutase, actin I, and cathepsin L cysteine proteinase, respectively (Tables 2 and 4). Predicted molecular mass and isoelectric point are shown in Tables 1, 2, 3, 4, and domains predicted by the SMART program and a National Center for Biotechnology Information (NCBI) conserved domain search are schematically shown in Fig. 2. Using the known amino acid sequences of ICP-1 [19], we performed local blast searches against sequence data obtained from this study. As a result, isotig 00046 was found to have a high sequence similarity with ICP-1 (Fig. 4). Gene expression analysis showed that ICP-1 gene is expressed in lophophore as well as in mantle tissues (Additional file 1). To estimate the abundance of each protein, we calculated the relative copy number based on the identified spectral counting [22–25]. The number of identified spectra of each protein was divided by the number of theoretically observable tryptic peptide ions, which have a mass-to-charge ratio of 400 to 1500 at two or three charge states, to generate an abundance index as the relative copy number. The result showed that isotig 00046 (ICP-1) is the most abundant protein in the shell extracts among the proteins identified in this study (Fig. 5). The percentage of spectra that were matched to peptide contained in protein sequences translated from transcriptome data was about 10 % of the total MS/MS spectra acquired. The proportion in this case was lower than those of other mass spectrometric analyses for organisms of which genome has been completely sequenced (S. cerevisiae, 40-50 %), suggesting presence of many more proteins in shell matrix samples than proteins identified in this study. Messenger RNA sequences in the transcriptome data may not include complete list for proteins in shell matrix or unknown chemical modifications that were not considered in our database search may occur in a large proportion of shell matrix proteins. The depth of isotigs is shown in Additional file 2.Fig. 1


Proteome analysis of shell matrix proteins in the brachiopod Laqueus rubellus.

Isowa Y, Sarashina I, Oshima K, Kito K, Hattori M, Endo K - Proteome Sci (2015)

Abundance index of shell matrix proteins identified in this study
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536745&req=5

Fig5: Abundance index of shell matrix proteins identified in this study
Mentions: SDS-PAGE of the soluble organic matrix extracted from the secondary layer showed two major bands (< 6.5 and 12 kDa) and a minor band of 62 kDa when stained with CBB (Fig. 1a). Silver staining showed a major band at 35 kDa and many minor bands in addition to the three bands stained with CBB (Fig. 1b). Transcriptome sequencing in mantle tissue generated a total of 125,437 reads. To identify shell matrix proteins from Laqueus rubellus, we performed a proteome analysis of the material from these different sources, namely (1) the soluble organic matrix from the whole shell, (2) insoluble organic matrix from the whole shell and (3) soluble organic matrix from the shell secondary layer. The generated MS/MS data were subjected to SEQUEST searches against the protein sequence database obtained from the transcriptome analysis. As a result, we identified 40 shell matrix proteins (Tables 1, 2, 3, 4 and Fig. 2). Among these proteins, 18 proteins were identified from the soluble organic matrix of the shell secondary layer, and the soluble and insoluble organic matrix from the whole shell (Table 1). While five proteins were identified from both the soluble and insoluble organic matrix of the whole shell, 17 proteins were only identified from either the soluble or insoluble organic matrices of the whole shell (Tables 2, 3, 4). A total of 22 proteins were deduced to be possibly complete sequences, because these sequences have a stop codon and either an in-frame start codon just after an in-frame stop codon in the 5′ region of the sequence or a potential signal peptide (Tables 1, 2, 3, 4). A blast search against the GenBank non-redundant database showed that 36 out of the 40 shell matrix proteins have no annotated homologous sequences. Among the proteins that have sequence homology, isotig 00281 showed relatively high sequence similarity with MSP130, which is a skeletal protein identified from sea urchins (Fig. 3) [20, 21]. Isotig 01587, isotig 00949, and isotig 00959 showed high sequence similarity with extracellular copper/zinc superoxide dismutase, actin I, and cathepsin L cysteine proteinase, respectively (Tables 2 and 4). Predicted molecular mass and isoelectric point are shown in Tables 1, 2, 3, 4, and domains predicted by the SMART program and a National Center for Biotechnology Information (NCBI) conserved domain search are schematically shown in Fig. 2. Using the known amino acid sequences of ICP-1 [19], we performed local blast searches against sequence data obtained from this study. As a result, isotig 00046 was found to have a high sequence similarity with ICP-1 (Fig. 4). Gene expression analysis showed that ICP-1 gene is expressed in lophophore as well as in mantle tissues (Additional file 1). To estimate the abundance of each protein, we calculated the relative copy number based on the identified spectral counting [22–25]. The number of identified spectra of each protein was divided by the number of theoretically observable tryptic peptide ions, which have a mass-to-charge ratio of 400 to 1500 at two or three charge states, to generate an abundance index as the relative copy number. The result showed that isotig 00046 (ICP-1) is the most abundant protein in the shell extracts among the proteins identified in this study (Fig. 5). The percentage of spectra that were matched to peptide contained in protein sequences translated from transcriptome data was about 10 % of the total MS/MS spectra acquired. The proportion in this case was lower than those of other mass spectrometric analyses for organisms of which genome has been completely sequenced (S. cerevisiae, 40-50 %), suggesting presence of many more proteins in shell matrix samples than proteins identified in this study. Messenger RNA sequences in the transcriptome data may not include complete list for proteins in shell matrix or unknown chemical modifications that were not considered in our database search may occur in a large proportion of shell matrix proteins. The depth of isotigs is shown in Additional file 2.Fig. 1

Bottom Line: We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins.The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences.Our study suggests an independent origin and unique mechanisms for brachiopod shell formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan.

ABSTRACT

Background: The calcitic brachipod shells contain proteins that play pivotal roles in shell formation and are important in understanding the evolution of biomineralization. Here, we performed a large-scale exploration of shell matrix proteins in the brachiopod Laqueus rubellus.

Results: A total of 40 proteins from the shell were identified. Apart from five proteins, i.e., ICP-1, MSP130, a cysteine protease, a superoxide dismutase, and actin, all other proteins identified had no homologues in public databases. Among these unknown proteins, one shell matrix protein was identified with a domain architecture that includes a NAD(P) binding domain, an ABC-type transport system, a transmembrane region, and an aspartic acid rich region, which has not been detected in other biominerals. We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins. The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences.

Conclusions: Our study suggests an independent origin and unique mechanisms for brachiopod shell formation.

No MeSH data available.