In-depth proteomic analysis of a mollusc shell: acid-soluble and acid-insoluble matrix of the limpet Lottia gigantea

Background

Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell.

Results

Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes.

Conclusions

The organic matrix of Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs.     

Structual comparison of dermatopontin amino acid sequences

Dermatopontin is a tyrosine-rich acidic extracellular matrix protein of 22 kD with possible functions in cellmatrix interactions and matrix assembly. Database of GenBank+EMBL+DDBJ sequences from Nucleotide, Gene, and Expressed Sequence Tag (EST) Divisions was searched with a keyword “dermatopontin” or mouse dermatopontin amino acid sequence. In addition to five mammals previously described, five mammalian, two bird, one fish dermatopontin genes were detected in vertebrates. Additionally, a goat EST was also shown as goat dermatopontin missing 5′-end of the coding region. Moreover, a mRNA sequence of rhesus monkey dermatopontin was identified, but the deduced amino acid sequence was terminated abruptly due to a nonsense codon. For three 6-residue repeat regions (D-R-E/Q-W-X-F/Y) that may function as part of a glycosaminoglycan binding site, the first repeat sequence is D-R-Q-W-N-Y in all mammals while Glutamine is substituted for Leucine in birds. The second and the third repeats are conserved in all vertebrates. The N-Y-D sequence, the consensus in many amine oxidases, is conserved in mammals except rodents. Asparagine is substituted for Threonine in birds. The tetrapeptide R-G-A-T sequence possibly recognizing the integrin family is conserved in mammals and birds, but Alanine was substituted for Glutamine in zebrafish resulting in loss of activity. In conclusion, functionally significant amino acid sequences in vertebrate dermatopontins are conserved in mammals, while they are not necessarily identified in birds and fish. The original function of vertebrate dermatopontins may be glycosaminoglycan binding and functions as a ligand for integrin and an amine oxidase may be gained in the process of evolution.     

Perlustrin, a Haliotis laevigata (abalone) nacre protein, is homologous to the insulin-like growth factor binding protein N-terminal module of vertebrates.

The 84-amino-acid-long sequence of perlustrin showed homology of the abalone nacre protein to the N-terminal domain of mammalian insulin-like growth factor binding proteins (IGFBPs). Despite the evolutionary distance between mollusks and mammals, the sequence identity was 40% including 12 conserved cysteines. However, the residues which were suggested recently to bind IGF-II in a complex with IGFBP-5 were conserved only partially. Nevertheless, perlustrin bound human IGFs with K(D) approximately 10(-7) M. This was the same affinity range as measured before for the interaction of isolated IGFBP-5 N-terminal domains with IGFs. Moreover, perlustrin bound bovine insulin with only approximately two- to sevenfold lower affinity than IGFs. Sequence similarity and growth factor binding identified perlustrin unequivocally as a member of the IGFBP family, the first found in an invertebrate biomineral. Nacre is known to contain proteinaceous factors which promote bone formation in vitro and in vivo. Bone contains IGFBPs which influence bone metabolism in many ways by modulating either IGF effects or IGF independently. Thus, perlustrin may provide a first clue at the molecular level to what these two phylogenetically rather distant biomineralization systems have in common.     

Complete cDNA sequence of SAP-like pentraxin from Limulus polyphemus: implications for pentraxin evolution

The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology.     

Spatio-temporal expression and regulation of dermatopontin in the early pregnant mouse uterus

During endometrial differentiation the extracellular matrix (ECM) changes dramatically to prepare for implantation of the embryo. However, the genes regulating the ECM build-up in the uterine endometrium during early pregnancy are not well known. Using the PCR-select cDNA subtraction method, dermatopontin was identified in the uterus of a pregnant mouse on day 4 of gestation. Dermatopontin mRNA increased dramatically on day 3, and was at its highest level at the time of implantation. Administration of RU 486 significantly inhibited mRNA expression by day 4 of gestation, but ICI 182,780 did not. Progesterone markedly induced dermatopontin expression in ovariectomized uteri within 4 h of administration, whereas estrogen had little effect. In silico analysis revealed progesterone receptor binding sites in the dermatopontin promoter region. Decidualization did not induce expression of dermatopontin; instead dermatopontin mRNA became strongly localized at the interimplantation site. In situ hybridization revealed that expression gradually decreased in the luminal epithelial cells as pregnancy progressed, whereas it increased in the stromal cells. The pattern of localization and the changes of intensity of dermatopontin mRNA coincided with those of collagen. Collectively, these results strongly suggest that dermatopontin expression is steroid-dependent. They also suggest that, at the time of implantation, dermatopontin expression is primarily regulated spatio-temporally by progesterone via progesterone receptors, and is modulated by the decidual response during implantation. Dermatopontin may be one of the regulators used to remodel the uterine ECM for pregnancy.     

Sequence diversification of the FK506-binding proteins in several different genomes.

Sequences of FK506-binding proteins (FKBPs) from four genomes of the following organisms were compared: the prokaryote Escherichia coli, the lower eukaryote Saccharomyces cerevisiae, the plant Arabidopsis thaliana, the nematode Caenorhabditis elegans and a composite of 14 unique FKBPs from two mammalian organisms Homo sapiens (man) and Mus musculus (domestic mouse). A singular FK506-like binding domain (FKBD) has about 12 kDa and occurs in the form of archetypal FKBP-12 and as a part of different proteins ranging in size from 13 to 135 kDa. Some organisms may contain a variable number of proteins which consist from two to four consecutively fused FKBDs. In the 12-kDa subgroup of archetypal FKBPs sequence identity (ID) varies from 100 to 83% (mammalian FKBPs-12), 75-50% in mammalian vs. invertebrate FKBPs-12, and fall to about 30% for pairwise sequence comparisons of mammalian and bacterial FKBPs-12 which suggests that their sequences are divergent. Multiple sequence alignment of FKBPs from the four genomes and a set of unique mammalian FKBPs does not contain any explicit consensus sequence but certain sequence positions have conserved physico-chemical characteristics. Variations of hydrophobicity and bulkiness in the multiple sequence alignment are nonsymmetrical because the physico-chemical properties of the aligned sequences changed during evolution. These variations at the sequence positions which are crucial for binding the immunosuppressive macrolide FK506 and peptidyl-prolyl cis/trans isomerase (PPIase) activity are small.     

miR-34基因家族的分子进化

根据miRNA基因在进化中高度保守的特点,利用生物信息学方法在目前已测序的动物物种中搜寻参与哺乳动物早期发育调控的mir-34基因的同源序列,在33个不同的动物物种中获得了miR-34基因的54条同源序列,其中18条为新发现的序列。表明miR-34是高度保守的,广泛存在于后生动物中。目前发现的mir-34基因80%位于基因间隔区,少数位于蛋白编码基因的内含子区和3′UTR上。不同动物中,mir-34基因成熟序列的同源性为68%,前体序列为38.89%。在无脊椎动物中只有一个mir-34,而在几乎所有的脊椎动物中都有mir-34a,mir-34b,mir-34c,形成miR-34基因家族。系统进化分析表明,脊椎动物中miR-34基因家族是通过基因的串联和局部重复形成的,这个过程中伴随着个别碱基的变异。     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.     

The Biomineralization Proteome: Protein Complexity for a Complex Bioceramic Assembly Process

There are over 62 different biominerals on Earth and a diverse array of organisms that generate these biominerals for survival. This review will introduce the process of biomineralization and the current understanding of the molecular mechanisms of mineral formation, and then comparatively explore the representative secretomes of two well‐documented skeletal systems: vertebrate bone (calcium phosphate) and invertebrate mollusk shell (calcium carbonate). It is found that both skeletal secretomes have gross similarities and possess proteins that fall into four functional categories: matrix formers, nucleation assisters, communicators, and remodelers. In many cases the mineral‐associated matrix former and nucleation assister sequences in both skeletal systems are unique and possess interactive conserved globular domains, intrinsic disorder, post‐translational modifications, sequence redundancy, and amyloid‐like aggregation‐prone sequences. Together, these molecular features create a protein‐based environment that facilitates mineral formation and organization and argue in favor of conserved features that evolve from the mollusk shell to bone. Interestingly, the mollusk shell secretome appears to be more complex compared to that of bone tissue, in that there are numerous protein subcategories that are required for the nucleation and organization of inner (nacre) and outer (prismatic) calcium carbonate regions of the shell. This may reflect the organizational and material requirements of an exoskeletal protective system.     

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.     

A collectin-like protein from tunicates

Collectins are a sub-family of C-type lectins from mammals and birds that are characterized by their collagen-like domains. The mammalian collectin, mannose binding lectin, has attracted considerable interest because it can activate complement components via a lectin-mediated complement pathway that is independent of immunoglobulins. In this study, we have identified a calcium-dependent lectin from the invertebrate (tunicate), Styela plicata, that bears substantial similarities to mammalian collectins. The tunicate lectin, which was isolated by carbohydrate affinity chromatography, has a reduced apparent molecular mass of 43 kDa. The 43 kDa reduced polypeptide appeared as dimers, trimers and hexamers when analyzed by non-reducing and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while gel filtration suggested that the native form of the protein was a nonamer. Amino acid sequence and amino acid composition analysis revealed obvious similarities between the tunicate lectin and mammalian collectins, notably the inclusion of a collagenous domain and a short, cysteine bearing N-terminal domain. The identification of a collectin-like protein in an invertebrate such as S. plicata, which does not express immunoglobulin, indicates that lectin-mediated complement pathways may predate the origin of antibodies.     

Molecular cloning of a chicken JAK homolog from activated T cells

A cDNA encoding a JAK-related protein was isolated from a chicken T cell library by screening with a PCR-generated probe that corresponds to a conserved region in the kinase domain. Sequence analysis reveals an ORF of 3318 nt, encoding a protein with a calculated molecular weight of 123 000. Chicken JAK (cJAK) contains a double catalytic domain that is characteristic of the JAK family of tyrosine kinases. Compared with mammalian JAKs, the kinase domain shows 70% sequence identity with the corresponding region of the mammalian JAKs. Overall, cJAK shows approximately 59% amino acid identity with mammalian JAK3s, and 52% amino acid identity with mammalian JAK2s. cJAK is expressed predominantly in thymus and spleen, with lower levels in kidney, thyroid and liver. cJAK is also expressed at low levels in unstimulated splenic T cells, whereas mRNA levels are increased after activation of the T cells with Con A. The sequence analysis and pattern of expression suggests that this is an avian homolog of JAK3.     

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.     

Molecular evolution of olfactomedin

Olfactomedin is a secreted polymeric glycoprotein of unknown function, originally discovered at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently found throughout the mammalian brain. As a first step toward elucidating the function of olfactomedin, its phylogenetic history was examined to identify conserved structural motifs. Such conserved motifs may have functional significance and provide targets for future mutagenesis studies aimed at establishing the function of this protein. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedins in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homopolymer formation such as the CXC motif near the amino terminus are conserved, as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, evolutionary analysis of a region of 53 amino acids of fish, frog, rat, mouse, and human olfactomedins indicates that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian, and mammalian lineages. Indeed, a distant olfactomedin homolog was identified in Caenorhabditis elegans. Although the amino acid sequence of this invertebrate protein is longer and highly divergent compared with its vertebrate homologs, the protein from C. elegans shows remarkable similarities in terms of conserved motifs and posttranslational modification sites. Six universally conserved motifs were identified, and five of these are clustered in the carboxyl terminal half of the protein. Sequence comparisons indicate that evolution of the N-terminal half of the molecule involved extensive insertions and deletions; the C-terminal segment evolved mostly through point mutations, at least during vertebrate evolution. The widespread occurrence of olfactomedin among vertebrates and invertebrates underscores the notion that this protein has a function of universal importance. Furthermore, extensive modification of its N-terminal half and the acquisition of a C-terminal SDEL endoplasmic-reticulum- targeting sequence may have enabled olfactomedin to adopt new functions in the mammalian central nervous system.      

Characterization of the clathrin heavy chain from Dictyostelium discoideum.

We report the cloning and analysis of a clathrin heavy-chain cDNA from the eukaryotic microorganism, Dictyostelium discoideum. A single gene, designated chcA, for the clathrin heavy chain encoded a protein of 1,694 amino acids with a molecular mass of 193,618 daltons. Comparison of the amino acid sequence with the rat and with the yeast sequence showed that the highly conserved protein was more similar to the mammalian clathrin heavy chain (57% identity) than to the yeast heavy chain (45% identity). The mRNA for the clathrin heavy chain was regulated during development. mRNA levels were highest during vegetative growth and declined as the cells progressed through the 24-hr developmental cycle. The concentration of clathrin heavy-chain protein was the same in cells grown in liquid media (high rates of pinocytosis) as in cells grown with bacteria (low rates of pinocytosis), which suggests that regulation of pinocytosis in these cells is not achieved by altering the concentration of clathrin.     

Glutamine synthetase II inRhizobium: Reexamination of the proposed horizontal transfer of DNA from eukaryotes to prokaryotes

Summary We have determined the DNA sequence of aRhizobium meliloti gene that encodes glutamine synthetase II (GSII). The deduced amino acid sequence was compared to that ofBradyrhizobium japonicum GSII and those of various plant and mammalian glutamine synthetases (GS) in order to evaluate a proposal that the gene for this enzyme was recently transferred from plants to their symbiotic bacteria. There is 83.6% identity between theR. meliloti andB. japonicum proteins. The bacterial GSII proteins average 42.5% identity with the plant GS proteins and 41.8% identity with their mammalian counterparts. The plant proteins average 53.7% identity with the mammalian proteins. Thus, the GS proteins are highly conserved and the divergence of these proteins is proportional to the phylogenetic divergence of the organisms from which the sequences were determined. No transfer of genes across large taxonomic gaps is needed to explain the presence of GSII in these bacteria.     

Characterization of two zebrafish cDNA clones encoding egg envelope proteins ZP2 and ZP3.

Two zebrafish cDNA clones encoding homologs of mammalian zona pellucida proteins ZP2 and ZP3 were isolated from a whole adult cDNA library. The ZP2 clone encodes a protein of 428 amino acids. Unlike other teleost ZP2s that contain an N-terminal repetitive domain enriched with prolines and glutamines, the zebrafish ZP2 has no such repetitive domain. In the C-terminal non-repetitive domain, the zebrafish ZP2 shares 55-76% sequence identity with other teleost ZP2s. The ZP3 cDNA clone encodes a protein of 431 amino acids, which shares 61% sequence identity with a carp ZP3. Similar to mammalian ZP proteins, both zebrafish ZP2 and ZP3 contain several potential phosphorylation sites. However, unlike mammalian ZP proteins, both zebrafish ZP proteins contain almost no glycosylation site, which has been proposed to be important for interaction with sperm; thus, the ZP proteins may behave differently in mammals and teleosts. Northern blot analysis indicated that both zebrafish ZP2 and ZP3 mRNAs were expressed exclusively in the ovary and hence the ovary is likely the only site for ZP2 and ZP3 biosynthesis.