Isolation and biochemical characterization of underwater adhesives from diatoms

Many aquatic organisms are able to colonize surfaces through the secretion of underwater adhesives. Diatoms are unicellular algae that have the capability to colonize any natural and man-made submerged surfaces. There is great technological interest in both mimicking and preventing diatom adhesion, yet the biomolecules responsible have so far remained unidentified. A new method for the isolation of diatom adhesive material is described and its amino acid and carbohydrate composition determined. The adhesive materials from two model diatoms show differences in their amino acid and carbohydrate compositions, but also share characteristic features including a high content of uronic acids, the predominance of hydrophilic amino acid residues, and the presence of 3,4-dihydroxyproline, an extremely rare amino acid. Proteins containing dihydroxyphenylalanine, which mediate underwater adhesion of mussels, are absent. The data on the composition of diatom adhesives are consistent with an adhesion mechanism based on complex coacervation of polyelectrolyte-like biomolecules.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Positive selection in the carbohydrate recognition domains of sea urchin sperm receptor for egg jelly (suREJ) proteins

A wealth of evidence shows that protein-carbohydrate recognition mediates the steps of gamete interaction during fertilization. Carbohydrate-recognition domains (CRDs) comprise a large family of ancient protein modules of approximately 120 amino acids, having the same protein fold, that bind terminal sugar residues on glycoproteins and polysaccharides. Sea urchin sperm express three suREJ (sea urchin receptor for egg jelly) proteins on their plasma membranes. suREJ1 has two CRDs, whereas suREJ2 and suREJ3 both have one CRD. suREJ1 binds the fucose sulfate polymer (FSP) of egg jelly to induce the sperm acrosome reaction. The structure of FSP is species specific. Therefore, the suREJ1 CRDs could encode molecular recognition between sperm and egg underlying the species-specific induction of the acrosome reaction. The functions of suREJ2 and suREJ3 have not been explored, but suREJ3 is exclusively localized on the plasma membrane over the sperm acrosomal vesicle and is physically associated with sea urchin polycystin-2, a known cation channel. An evolutionary analysis of these four CRDs was performed for six sea urchin species. Phylogenetic analysis shows that these CRDs were already differentiated in the common ancestor of these six sea urchins. The CRD phylogeny agrees with previous work on these species based on one nuclear gene and several mitochondrial genes. Maximum likelihood shows that positive selection acts on these four CRDs. Threading the suREJ CRDs onto the prototypic CRD crystal structure shows that many of the sites under positive selection are on extended loops, which are involved in saccharide binding. This is the first demonstration of positive selection in CRDs and is another example of positive selection acting on the evolution of gamete-recognition proteins.     

Characterization of a cDNA clone coding for a sea urchin histone H2A variant related to the H2A.F/Z histone protein in vertebrates.

A cDNA clone coding for a sea urchin histone H2A variant has been isolated. The coding region of the clone has been sequenced and the sequence found to be closely related to the H2A.F sequence in chickens. The nucleotide sequence of the sea urchin H2A.F/Z is 74% conserved when compared to chicken H2A.F and 51% conserved compared to sea urchin H2A early and 60% compared to sea urchin H2A late. The nucleotide-derived amino acid comparisons show that H2A.F/Z is 97% homologous with H2A.F in chickens and 57% and 56% homologous when compared to sea urchin H2A early and late respectively. There are between 3-6 copies of the H2A.F/Z sequence in the S. purpuratus genome. The H2A.F/Z gene sequence codes for the previously identified H2A.Z protein. All embryonic stages and adult tissues tested contain mRNA for H2A.F/Z. The mRNA appears in the poly A+ RNA fraction after chromatography over oligo dT cellulose.     

Pulcherosine, a novel tyrosine-derived, trivalent cross-linking amino acid from the fertilization envelope of sea urchin embryo

The normally hardened and aminotriazole-induced soft fertilization envelopes (FEs) of the sea urchin Hemicentrotus pulcherrimus and two other species were isolated and investigated for component proteins and cross-linking amino acids. From the acid hydrolysate of the hard FE of H. pulcherrimus, we isolated by reversed-phase high-performance liquid chromatography a novel fluorescent compound as well as dityrosine and trityrosine, the major tyrosine-derived cross-linking amino acids. These three compounds were also isolated from the reaction products of the tyrosine/horseradish peroxidase/H2O2 system. The structure of the novel compound, designated "pulcherosine", was determined to be 5-[4"-(2-carboxy-2-aminoethyl)phenoxy]-3,3'-dityrosine. With respect to the position of diphenyl ether bond between the tyrosine and dityrosine moieties, it is an isomer of isotrityrosine found in Ascaris cuticle collagen [Fujimoto et al. (1981) Biochem. Biophys. Res. Commun. 99, 637-643]. Isotrityrosine was not found in either of the above systems as a major component. The contents of tyrosine, dityrosine, trityrosine, and pulcherosine in the hard FE of H. pulcherrimus were estimated as 255, 5.5, 2.1, and 1.3 residues, respectively, per 10,000 total amino acid residues, while in the soft FE, those of tyrosine and dityrosine were 305 and 0.25 residues, respectively, and trityrosine and pulcherosine were only traces. The molar ratio of dityrosine, trityrosine, and pulcherosine in the hard FE was 100:38:24, while that for tyrosine/horseradish peroxidase/H2O2 reaction products was 100:3:8, respectively.     

SPXX, a frequent sequence motif in gene regulatory proteins

A new DNA-binding unit, composed of four amino acid residues and common in gene regulatory proteins, is proposed. The occurrences of the sequences Ser-Pro-X-X (SPXX) and Thr-Pro-X-X (TPXX) in gene regulatory proteins are compared with those in general proteins. These sequences are found more frequently in gene regulatory proteins including homoeotic gene products, segmentation gene products, steroid hormone receptors and certain oncogene products, than they are in DNA-binding proteins that are not directly involved in gene regulation, such as the core histones, or in general proteins. It is therefore suggested that these sequences contribute to DNA-binding in a manner important for gene regulation. Amino acid residues characteristic of the types of proteins are found as the variable residues X: basic residues, Lys and Arg, in histones, H1 and sea urchin spermatogenous H2B; Tyr in RNA polymerase II; and Ser, Thr, Ala, Leu and Pro in other gene regulatory proteins S(T)PXX sequences are located on either side of other DNA-recognizing units such as Zn fingers, helix-turn-helices, and cores of histones. The structure of a S(T)PXX sequence is presumed to be a beta-turn I stabilized by two hydrogen bonds, and its potential mode of DNA-binding is discussed.     

A new hyperpolarization-activated, cyclic nucleotide-gated channel from sea urchin sperm flagella

A sea urchin sperm flagellar hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel is known (SpHCN1) that is modulated by cAMP. Here, we describe a second flagellar HCN channel (SpHCN2) cloned from the same sea urchin species. SpHCN2 is 638 amino acids compared to 767 for SpHCN1. SpHCN2 has all the domains of an HCN channel, including six transmembrane segments (S1-S6), the ion pore, and the cyclic nucleotide-binding domain. The two full-length proteins are 33% identical and 51% similar. The six transmembrane segments vary from 46-79% identity. S4, which is the voltage sensor, is 79% identical between the two proteins. The ion selectivity filter sequence is GYG in the ion pore of SpHCN1 and GFG in SpHCN2. By sequence, SpHCN2 is 73.5kDa, but it migrates on SDS-PAGE at 64kDa. Western immunoblots show localization to flagella, which is confirmed by immunofluorescence. A neighbor-joining tree shows that SpHCN2 is basal to all known HCN channels. SpHCN2 might be the simplest pacemaker channel yet discovered.     

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.     

A histone H2B variant from the embryo of the sea urchin Parenchinus angulosus

A variant of histone H2B has been isolated from sea urchin embryo (Parenchinus angulosus). Out of the 53 amino acids positioned in the three CNBr-peptides only 26 residues are identical to those in the corresponding positions of calf thymus histone H2B. A similar degree of homology exists between the embryonic variant and the previously characterized variants from sperm cells of the same organism.     

Is the adhesive material secreted by sea urchin tube feet species‐specific?

Sea urchin adoral tube feet are highly specialized organs that have evolved to provide efficient attachment to the substratum. They consist of a disk and a stem that together form a functional unit. Tube foot disk tenacity (adhesive force per unit area) and stem mechanical properties (e.g., stiffness) vary between species but are apparently not correlated with sea urchin taxa or habitats. Moreover, ultrastructural studies of sea urchin disk epidermis pointed out differences in the internal organization of the adhesive secretory granules among species. This prompted us to look for interspecific variability in the composition of echinoid adhesive secretions, which could explain the observed variability in adhesive granule ultrastructure and disk tenacity. Antisera raised against the footprint material of Sphaerechinus granularis (S. granularis) were first used to locate the origin of adhesive footprint constituents in tube feet by taking advantage of the polyclonal character of the generated antibodies. Immunohistochemical assays showed that the antibodies specifically labeled the adhesive secretory cells of the disk epidermis in the tube feet of S. granularis. The antibodies were then used on tube foot histological sections from seven other sea urchin species to shed some light on the variability of their adhesive substances by looking for antibody cross‐reactivity. Surprisingly, no labeling was observed in any of the species tested. These results indicate that unlike the adhesive secretions of asteroids, those of echinoids do not share common epitopes on their constituents and thus would be “species‐specific.” In sea urchins, variations in the composition of adhesive secretions could therefore explain interspecific differences in disk tenacity and in adhesive granule ultrastructure. J. Morphol., 2011. © 2011 Wiley Periodicals, Inc.     

Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin (Strongylocentrotus intermedius) ovaries

Fasciclin I, a neuronal cell adhesion molecule, was first identified in the grasshopper. To date, various fasciclin I-like proteins have been identified but their biological functions have not been well characterized. Here, we have purified a fasciclin I-like protein with a molecular weight of 33kDa from sea urchin (Strongylocentrotus intermedius) ovaries using hydrophobic chromatography and gel filtration. The protein was not N-glycosylated. Partial amino acid sequences of cyanogen bromide (CNBr)-cleaved fragments were highly conserved to other sea urchin fasciclin I-like proteins identified previously. The circular dichroism (CD) spectrum analysis demonstrated that the 33kDa protein contained high content of alpha-helical structure. These results suggest that the 33kDa protein is a fasciclin I-like family. Additionally, the fasciclin I-like protein promoted HT1080 human fibrosarcoma cell attachment. Further, a synthetic peptide (P1: GLREAANIAEQVDLRQVLRDVDL) of the protein corresponding to a highly conserved region of the fasciclin I-like family promoted heparin-dependent HT1080 cell attachment. Moreover, the peptide inhibited HT1080 cell attachment to the fasciclin I-like protein. These results suggest that the 33kDa protein from sea urchin ovaries isolated here is a member of the fasciclin I family and that the N-terminal region of the protein is important for cell attachment activity. The protein has a potential to be involved in biological functions in sea urchin as a cell adhesive molecule.     

Cloning of a sea urchin sarco/endoplasmic reticulum Ca2+ ATPase

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), a vesicular integral membrane protein, is the best-characterized member of the P-type ion translocating ATPase superfamily. Here we describe the cloning and structural analysis of a sea urchin SERCA (suSERCA) cloned from testis cDNA. The approximately 112 kDa suSERCA is 1022 amino acids with approximately 70% identity and 80% similarity to all known mammalian SERCA isoforms. suSERCA shares all the structural features of mammalian SERCAs, including domains: A, actuator; N, nucleotide-binding; and P, phosphorylation, and also 10 transmembrane helices. Like human SERCA2, the suSERCA has a possible 11th transmembrane segment in its extreme C-terminus. The alignment of three sequences (suSERCA, human SERCA2, and rabbit SERCA1a) shows that the Ca2+ binding residues and kinks (required to form the ion-binding pocket) are 100% conserved. The annotated suSERCA gene consists of 24 exons separated by 23 introns and is approximately 30 kb. Western blots show that suSERCA is present in sea urchin eggs and testis, but not in mature spermatozoa. Treatment of live sperm with SERCA inhibitors has no effect on intracellular calcium, suggesting the absence of SERCA in sea urchin spermatozoa.     

Studies on protein synthesis during sea urchin oogenesis. I. Synthesis of histone F2b

Basic proteins were extracted from sea urchin oocytes previously incubated with 3H-lysine and then were analyzed by electrophoresis. A very radioactive band, which showed the same mobility as histone F2b, was analyzed for its amino acid composition. The results show an identity between this protein and histones F2b. In addition, an improved method of isolating large amounts of sea urchin oocytes is described.     

Separation and properties of an H2B histone variant from the sperm chromatin of the sea urchin Sphaerechinus granularis

The purification and the physico-chemical characterization of one of the two H2B histone variants from the sperm of the sea urchin Sphaerechinus granularis are reported. The molecule shows, in addition to a distinctive molecular weight value, an amino acid composition different both from that of calf thymus H2B histone and from those of H2B histones from chromatin of sperm and embryos of other sea urchins. Circular dichroism and fluorescence data are discussed in comparison to those of calf thymus H2B.     

Opsins and clusters of sensory G-protein-coupled receptors in the sea urchin genome

Rhodopsin-type G-protein-coupled receptors (GPCRs) contribute the majority of sensory receptors in vertebrates. With 979 members, they form the largest GPCR family in the sequenced sea urchin genome, constituting more than 3% of all predicted genes. The sea urchin genome encodes at least six Opsin proteins. Of these, one rhabdomeric, one ciliary and two G(o)-type Opsins can be assigned to ancient bilaterian Opsin subfamilies. Moreover, we identified four greatly expanded subfamilies of rhodopsin-type GPCRs that we call sea urchin specific rapidly expanded lineages of GPCRs (surreal-GPCRs). Our analysis of two of these groups revealed genomic clustering and single-exon gene structures similar to the most expanded group of vertebrate rhodopsin-type GPCRs, the olfactory receptors. We hypothesize that these genes arose by rapid duplication in the echinoid lineage and act as chemosensory receptors of the animal. In support of this, group B surreal-GPCRs are most prominently expressed in distinct classes of pedicellariae and tube feet of the adult sea urchin, structures that have previously been shown to react to chemical stimuli and to harbor sensory neurons in echinoderms. Notably, these structures also express different opsins, indicating that sea urchins possess an intricate molecular set-up to sense their environment.     

Mapping of magnesium and of different protein fragments in sea urchin teeth via secondary ion mass spectroscopy

Mature portions of sea urchin are comprised of a complex array of reinforcing elements yet are single crystals of high and very high Mg calcite. How a relatively poor structural material (calcite) can produce mechanically competent structures is of great interest. In teeth of the sea urchin Lytechinus variegatus, we recorded high-resolution secondary ion mass spectrometry (SIMS) maps of Mg, Ca ,and specific amino acid fragments of mineral-related proteins including aspartic acid (Asp). SIMS revealed strong colocalization of Asp residues with very high Mg. Demineralized specimens showed serine localization on membranes between crystal elements and reduced Mg and aspartic acid signals, further emphasizing colocalization of very high Mg with ready soluble Asp-rich protein(s). The association of Asp with nonequilibrium, very high magnesium calcite provides insight to the makeup of the macromolecules involved in the growth of two different composition calcites and the fundamental process of biomineralization.     

Solid residues from<Emphasis Type="Italic"> Ruminococcus</Emphasis> cellulose fermentations as components of wood adhesive formulations

Residues from the fermentation of cellulose by the anaerobic bacteria Ruminococcus albus (strain 7) or Ruminococcus flavefaciens (strains FD-1 or B34b) containing residual cellulose, bacterial cells and their associated adhesins, were examined for their ability to serve as components of adhesives for plywood fabrication. The residues contained differing amounts of protein (0.4–4.2% of dry weight), but the ratios of monosaccharides recovered following two-stage treatment of the residue with detergent (pH 7) and TFA were similar for all three strains (0.71 glucose:0.18 xylose:0.08 mannose:0.02 galactose), suggesting similarities in exopolysaccharide composition. Three-ply aspen panels prepared with fermentation residues (FR) displayed better shear strength and wood failure under dry conditions than following a vacuum/pressure/soak/dry treatment, but adhesive properties were inferior to those prepared with conventional phenol-formaldehyde (PF) adhesives. However, panels prepared by incorporating the R. albus 7 FR into PF formulation, at 73% by weight of the total adhesive, exhibited shear strength and wood failure similar to that obtained with PF adhesive alone. Use of residues from fermentations by these bacteria as components of adhesives may add value to biomass fermentations aimed primarily at producing ethanol and other chemical products.Mention of specific products is for informational purposes only and does not imply an endorsement or warranty by the United States Department of Agriculture, over similar products that may also be suitable.     

The phylogeny and chemical diversity of quinone-tanned glues and varnishes

1. 3,4-Dihydroxyphenyl-L-alanine (DOPA)-containing proteins are widely distributed throughout the animal kingdom and appear to serve chiefly as waterproof adhesives and varnishes. 2. The unique chemical and physical stability of these adhesives and varnishes is imparted by quinone-tanning, an oxidative process that leads to the polymerization of DOPA-containing and other proteins. 3. Recent advances in the biochemistry of DOPA-containing proteins suggest that most consist of tandemly repeated sequence motifs. Each motif contains DOPA, a basic amino acid (usually lysine), and abundant glycine or proline. 4. The DOPA residues undergo catechol oxidase-catalyzed conversion to o-quinones at the onset of quinone-tanning. 5. The complexity of quinone chemistry is discussed with regard to quinone-tanning.