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J H Waite

Publications and source records attributed to J H Waite.

At least 19 recordsLinked to original sources
PubMed1998

Structure and mucoadhesion of mussel glue protein in dilute solution.

Purified mussel adhesive protein mefp-1 (Mytilus edulis foot protein 1) has been studied regarding its state of oligomerization and gross conformation in dilute solution. Sedimentation equilibrium in the analytical ultracentrifuge of a dilute solution of protein (0.4 mg/mL) in acetate buffer at pH 4.5 and I = 0.10 M yielded an apparent molecular weight (whole distribution weight average, Mw, app) of 114 000 +/- 5000 via the "M" procedure, a value in almost exact agreement with the monomeric molecular weight obtained by MALDI mass spectrometry. At this low concentration, it is reasonable to assume thermodynamic ideality, i.e., Mw,app approximately Mw. This result, together with plots of point weight average apparent molecular weight versus concentration for three different loading concentrations (0.4, 0.8, 1.0 mg/mL), clearly demonstrates that this protein is essentially monomeric in dilute solution. Sedimentation velocity experiments yielded an estimate of the sedimentation coefficient s020,w = 2.34 +/- 0.17 S, which for M = 110 000 gives a frictional ratio f/f0 = 3.2 +/- 0.3. The interpretation of this, in terms of an extended rather than globular conformation for the structure of mefp-1 in dilute solution, is considered, within plausible limits of molecular hydration, and models for the structure in solution are considered, in light of the thermodynamic nonideality behavior of these molecules and previously published circular dichroism data. The significance of these observations in terms of the bioadhesive properties of mefp-1 is described, and the very strong interaction in dilute solution with a mucin glycoprotein is demonstrated.

Adhesiveness↗
PubMed1998

A potential mediator of collagenous block copolymer gradients in mussel byssal threads.

Mussel byssal threads contain unusual block copolymer-like proteins that combine collagen with flanking domains that resemble silk-fibroin (preCol-D) or elastin (preCol-P). These are distributed in complementary gradients along the length of the threads and as precursors in the mussel foot. We discuss a 76-kDa precursor, preCol-NG, from a cDNA library of the foot where it has no gradient but rather is distributed evenly along the distal to proximal axis. A pepsin-resistant fragment of preCol-NG has been confirmed in byssal threads. Like preCol-D and -P, this protein has a central collagenous domain, flanking domains, an acidic patch, and histidine-rich termini. The flanking domains of preCol-NG resemble the glycine-rich proteins of plant cell walls with tandem XGlyn repeats where X denotes alanine, leucine, or asparagine but not proline. Similarity with the (glycine-alanine) repeats and poly(alanine) runs of arthropod silks also exists. Based on available evidence, a model of preCol axial assembly is proposed in which preCol-NG functions as a mediator between preCol-D/-P molecules. This is consistent with the observed progression of mechanical properties in byssal threads.

Amino Acid Sequence↗
PubMed1998

The peculiar collagens of mussel byssus.

The byssal collagens of marine mussels are extracorporeal collagens that function in byssal threads under tension. Each byssal thread resembles a shock absorber in its mechanical design: it is strong and stiff at one end and pliably elastic at the other. Primary structures of three of these collagens (preCols), deduced from cDNAs, reveal signal peptide sequences, but no N-glycosylation sites or propeptides typical of procollagens. The collagen domain (40-50 kDa) represents roughly half the mass of the mature molecules and is distinguished by its central location, abundant Gly-Gly-X repeats, and "flaws" (usually Gly deletions). Flanking the collagen domains on both sides are structural domains that resemble elastin in preCol-P, spider drag-line silk in preCol-D, and Gly-rich cell wall proteins in preCol-NG. Not surprisingly, studies of preCol distribution in byssal threads suggest preCol-P enhancement in the elastic proximal portion, while preCol-D predominates in the stiffer distal portion. PreCol-NG, in contrast, is evenly distributed. Although no data are yet available on the fibrillogenesis and cross-linking of the preCols, the quarter-stagger assembly of fibrillar interstitial collagens does not pertain since preCols lack the terminal peptides of tropocollagen. Metal-binding by histidines may mediate the initial inter- and intramolecular stabilization of preCols in the byssus.

Animals↗
PubMed1998

Use of methacrylate De-embedding protocols for in situ hybridization on semithin plastic sections with multiple detection strategies.

New plastic resins are gradually replacing traditional paraffin-embedding in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microscopic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically increasing hybridization signal compared to that obtained in other plastics. The goal of this study was to evaluate the general applicability of the methacrylate embedding acetone de-embedding (MEADE) technique for ISH investigations. Several high-resolution, high-sensitivity ISH protocols are described, using both end-labeled oligonucleotides and randomly primed DNA probes (200-400 BPS), signal amplification by catalyzed reporter deposition (CARD), and chromogenic and fluorescent detection methods. With slight modification, the MEADE ISH technique permitted localization of bacterial symbionts in fragile gill tissue and collagen transcripts in foot tissue of two marine bivalves. MEADE ISH has proved extremely versatile and will likely be suitable for many future applications.

Animals↗
PubMed1998

A major protein precursor of zebra mussel (Dreissena polymorpha) byssus: deduced sequence and significance.

The zebra mussel is a nonindigenous invader of North American lakes and rivers and one of the few freshwater bivalve molluscs having a byssus--a sclerotized organ used by the mussel for opportunistic attachment to hard surfaces. We have sequenced a foot-specific cDNA whose composite protein sequence was deduced from a series of overlapping but occasionally nonidentical cDNA fragments. The overall deduced sequence matches tryptic peptides from a major byssal precursor protein--Dreissena polymorpha foot protein 1 (Dpfp1). The calculated mass of Dpfp1 is 49 kDa; but this is known to be extensively hydroxylated and O-glycosylated during maturation. Purified native Dpfp1 analyzed using matrix-assisted laser-desorption ionization mass spectrometry with time-of-flight indicates that the protein occurs as at least two size variants with masses of 48.6 and 54.5 kDa. In all probability, the sequence variants reported in this study are related to the larger mass variant. Dpfp1 has a block copolymer-like structure defined by two consensus motifs that are sharply segregated into domains. The N-terminal side of Dpfp1 has 22 tandem repeats of a heptapeptide consensus (P-[V/E]-Y-P-[T/S/delta]-[K/Q]-X); the C-terminal side has 16 repeats of a tridecapeptide motif (K-P-G-P-Y-D-Y-D-G-P-Y-D-K). Both consensus repeats are unique, with some limited homology to other proteins functioning in tension: marine mussel adhesives, plant extensins, titin, and trematode eggshell precursors.

Amino Acid Sequence↗
PubMed1997

Tough tendons. Mussel byssus has collagen with silk-like domains.

The primary structure of the alpha-chain of preCol-D (molecular mass = 80 kDa), a tanned collagenous protein predominating in the distal portion of the byssal threads of the mussel Mytilus edulis, was deduced from cDNA to encode an unprecedented natural block copolymer with three major domain types: a central collagen domain flanked by fibroin-like domains and followed by histidine-rich termini. The fibroin-like domains have sequence motifs that strongly resemble the crystalline polyalanine-rich and amorphous glycine-rich regions of spider dragline silk fibroins. The terminal regions resemble the histidine-rich domains of a variety of metal-binding proteins. The silk domains may toughen the collagen by increasing its strength and extensibility. PreCol-D expression is limited to the mussel foot, which contains a longitudinal gradient of preCol-D mRNA. This gradient increases linearly in the proximal to distal direction and reaches a maximum just before the distal depression of the foot.

Amino Acid Sequence↗
PubMed1997

Extensible collagen in mussel byssus: a natural block copolymer.

To adhere to solid surfaces, marine mussels produce byssal threads, each of which is a stiff tether at one end and a shock absorber with 160 percent extensibility at the other end. The elastic extensibility of proximal byssus is extraordinary given its construction of collagen and the limited extension (less than 10 percent) of most collagenous materials. From the complementary DNA, we deduced that the primary structure of a collagenous protein (preCol-P) predominating in the extensible proximal portion of the threads encodes an unprecedented natural block copolymer with three major domain types: a central collagen domain, flanking elastic domains, and histidine-rich terminal domains. The elastic domains have sequence motifs that strongly resemble those of elastin and the amorphous glycine-rich regions of spider silk fibroins. Byssal thread extensibility may be imparted by the elastic domains of preCol-P.

Alanine↗
PubMed1996

In situ analysis of peptidyl DOPA in mussel byssus using rotational-echo double-resonance NMR.

Rotational-echo double-resonance (REDOR) 13C NMR spectra with 2H dephasing have been obtained from plaques and threads from the byssus of the marine mussel Mytilus edulis labeled by sea-water exposure to L-[ring-4-(13)C]tyrosine and L-[ring-d4]tyrosine for 2 days. Specific isotopic enrichment of tyrosine in protein reached 25% in both 13C and 2H. Fifteen percent of the total 13C label was incorporated as diphenolic carbon. Based on REDOR dephasing, about one-tenth of tyrosine rings in both intact plaques and threads are within 4 A of each other or rings of 3,4-dihydroxy-phenylalanine (DOPA). However, there is no direct evidence for the formation of covalent linkages between or among tyrosine and DOPA rings in either plaques or threads.

Animals↗
PubMed1996

Cloning, sequencing and sites of expression of genes for the hydroxyarginine-containing adhesive-plaque protein of the mussel Mytilus galloprovincialis.

A segment of Mytilus galloprovincialis foot protein 3 (Mgfp-3) cDNA was amplified by means of reverse-transcription (RT)/PCR with degenerate primers. The 5' and 3' regions of the cloned segment were amplified by means of rapid amplification of cDNA ends. The 5'-region clones had almost identical nucleotide sequences, but two sequences were found among 3'-region clones. The Mgfp-3 coding region was amplified between a 5' untranslated sequence and one of two 3' untranslated sequences. Two cDNA clones which encoded variants Mgfp-3A and Mgfp-3B, were isolated. These two clones encoded proteins with 70 and 77 amino acid residues, of which the first 24 residues are predicted to be signal peptides. The existence of additional variants was suggested by the sequences of other clones. Thus, it was suggested that Mgfp-3 genes constitute a gene family. RT/PCR of RNA from developing larvae indicates that Mgfp-3 genes are transcribed after settlement. RT/PCR of RNA from major organs and in situ hybridization of the foot indicate that Mgfp-3 genes are transcribed in a limited part of the foot, i.e., the phenol gland and a distal part of the accessory gland.

Amino Acid Sequence↗
PubMed1995

Novel 3,4-di- and 3,4,5-trihydroxyphenylalanine-containing polypeptides from the blood cells of the ascidians Ascidia ceratodes and Molgula manhattensis.

Acetic acid urea extraction of the blood cells of two ascidian species yielded four novel families of polypeptides (3500-5300 Da) containing 3,4-dihydroxyphenylalanine (DOPA) and 3,4,5-trihydroxyphenylalanine (TOPA) from the Phlebobranch Ascidia ceratodes and two DOPA proteins (9-10 kDa) from the Stolidobranch Molgula manhattensis. 3,4,5-Trihydroxyphenylalanine residues have not been found previously in polypeptides in any biological system. The DOPA content of the M. manhattensis proteins is the highest yet reported for a naturally occurring DOPA protein. The successful isolation of proteinaceous components from A. ceratodes blood cells requires the incorporation of high concentrations of complexing agent in the extraction buffers to inactivate vanadium(III) which forms intractable organometallic polymers. The A. ceratodes blood cell polypeptides are all rich in alanine and TOPA residues, have neutral to basic pI values, and, while all give single bands on acid urea-polyacrylamide electrophoresis, they exhibit extensive microheterogeneity. This is reflected by their large molecular weight distribution as determined by matrix-assisted laser desorption ionization-mass spectrometry, the variation in the ratio of their 280-nm absorption to chromophores of unknown structure in the polypeptide's electronic absorption spectra, and the N-terminal sequence analysis. The two M. manhattensis proteins consist largely of four amino acids (alanine, valine, phenylalanine, and DOPA) with DOPA accounting for upward of 40 mol%. Both give an N-terminus of Ala-Phe-Tyr before resisting further progress by Edman degradation. Proteins and polypeptides from both ascidians are extremely resistant to proteases, a property which, while hampering characterization by sequence analysis, appears ideally suited to their proposed function of forming an impervious hemostat at the site of vascular injury. The yield of proteins and polypeptides relative to tunichromes appears to be seasonally dependent. The presence of DOPA and TOPA moieties in the proteins and polypeptides implicates them, as well as the tunichromes, as potential metal-sequestering agents.

Amino Acid Sequence↗
PubMed1995

Hydroxyarginine-containing polyphenolic proteins in the adhesive plaques of the marine mussel Mytilus edulis.

An unusual polymorphic protein family of nine or more variants has been isolated from the byssal adhesive plaques and foot of the marine mussel Mytilus edulis. In accordance with established terminology, the family is referred to as M. edulis foot protein 3 or simply Mefp-3. Variants of Mefp-3 have molecular masses of about 6 kDa, isoelectric points greater than 10.5, and an amino acid composition dominated by six amino acids: glycine, asparagine, 3,4-dihydroxyphenylalanine (Dopa), tryptophan, arginine, and an unknown basic amino acid. The latter has been isolated and identified as 4-hydroxyarginine using fast atom bombardment mass spectrometry and appropriate standards. The primary structure of variant Mefp-3F has been determined by peptide mapping using automated Edman sequencing in combination with fast atom bombardment and matrix-assisted laser desorption ionization mass spectrometry: ADYYGPNYGPPRRYGGGNYNRYNRYGRRYGGYKGWNNGWNRGRRGKYW where Y represents Dopa, and R represents hydroxyarginine. Notably, the 4 occurrences of RY are marked by a resistance to trypsin digestion. Although the conversion of tyrosines to Dopa is essentially complete, hydroxylation of arginines varies between 40 and 80%. In contrast to other mussel adhesive proteins such as Mefp-1 and -2 which have large numbers of highly conserved, tandemly repeated peptide motifs, Mefp-3 has only short sporadic repeats. The specific function of Mefp-3 in byssal adhesion is unknown.

Amino Acid Sequence↗
PubMed1995

Exotic collagen gradients in the byssus of the mussel Mytilus edulis.

Byssal threads of the common mussel Mytilus edulis contain collagenous molecules from which two pepsin-resistant fragments have been isolated and characterized. These show a complementary distribution along the length of the thread, such that one predominates distally (Col-D) and the other proximally (Col-P). Both fragments contain three identical alpha-like chains with molecular masses of 50 kDa (Col-P) and 60 kDa (Col-D) and have typically collagenous amino acid compositions; for example, 35% glycine and almost 20% proline plus 4-trans-hydroxyproline. Hydroxylysine and 3-hydroxyproline were absent. Col-P sequences are also typical of collagen in consisting of tandem repeats of the triplet Gly-X-Y in which X and Y generally represent any amino acid. When proline occurs, it is hydroxylated to 4-trans-hydroxyproline only in the Y position. Seven instances where X is glycine have been detected in Col-P. Specific polyclonal anti-Col antibodies were used to isolate the precursors of Col-P and Col-D from the mussel foot. PreCol-P has a molecular mass of 95 kDa and contains 36% glycine but a lower imino acid content (13%). It has a complementary distribution with another precursor (preCol-D, 97 kDa) along the length of the foot. The two precursor compositions suggest resilin-like and silk-fibroin-like structures, respectively, in the noncollagenous domains of preCol-P and preCol-D. Immunogold labelling studies indicate that Col-P is associated with the coiled fibers of the inner core in the proximal portion of the thread, whereas Col-D is localized to the straight fiber bundles of the distal thread as well as to the outer core of the proximal thread.

Amino Acid Sequence↗
PubMed1995

Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus.

Variation in the adhesive protein gene sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus collected in Delaware, Kamaishi (Japan), and Alaska, respectively, was analyzed by the polymerase chain reaction (PCR) using two sets of oligonucleotide primers. The first set, Me 13 and Me 14, was designed to amplify the repetitive region. The length of the amplified fragments was highly variable, even among samples of the same species. Another set, Me 15 and Me 16, was designed to amplify a part of the nonrepetitive region. The length of the amplified fragments was uniform in each species and differed interspecifically; 180, 168, and 126 bp for M. edulis, M. trossulus, and M. galloprovincialis, respectively. The amplified sequence of M. trossulus resembled that of M. edulis. Mussels from other sites were also examined by PCR using Me 15 and Me 16. Wild mussels from Tromsö (Norway) and cultured mussels from Brittany (France) were identified as M. edulis. Cultured mussels from the Mediterranean coast of France and wild mussels from Shimizu (Japan) were identified as M. galloprovincialis. Some wild mussels from Hiura (Japan) were identified as a hybrid between M. galloprovincialis and M. trossulus. Thus, the length of this part (variable region) of the sequence is proposed as a diagnostic marker for these three morphologically similar species and their hybrids.

Amino Acid Sequence↗
PubMed1995

Wresting the muscle from mussel beards: research and applications.

Marine and zebra mussels secrete byssal beards to attach themselves opportunistically to hard surfaces in their environment. By doing this, they naturally earn a reputation as fouling pests. The protein precursors of byssus in mussels are being investigated in the hope not only of discovering specific measures against these marine foulers, but also to gain some insights into the technically challenging task of engineering adhesive bonds underwater. Although byssal proteins are all part of the bearded glue that bonds them to a surface, they can be subdivided into three types depending on the function that they serve in byssal threads: (1) fibrous proteins form the load-bearing cables in the core of the threads, (2) cuticular proteins form a protective coat around the cables, and (3) adhesive proteins connect the cables to a foreign surface. A flaw in any one of these will undermine a mussel's ability to attach. The fibrous proteins can be collagenous, silk-like, elastic, or any combination of these. Covering these are the cuticular proteins, which are distinguished by their surface coupling properties, tandemly repeated primary sequence, and their high content of lysine and the exotic amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA). The adhesive proteins are of low molecular weight, contain DOPA, and assemble to form microcellular solids (foams). Several of these proteins are already attracting biotechnological attention as cell and tissue attachment factors, anticorrosives, and metal-sequestering reagents.

Adhesives↗
PubMed1993

Eggshell formation in Bdelloura candida, an ectoparasitic turbellarian of the horseshoe crab Limulus polyphemus.

The marine triclad Bdelloura candida (Turbellaria) episodically deposits stalked eggshells onto the gill lamellae of the horseshoe crab Limulus polyphemus. Ultrastructural and biochemical analyses indicate that the eggshells consist of primary inner and secondary outer layers. Protein of the primary layer is rich in glycine, aspartate/asparagine, serine, and 3,4-dihydroxyphenylalanine (Dopa). In this regard it resembles the eggshell compositions of other members of the phylum Platyhelminthes. The primary layer appears to be derived from precursors produced in the vitelline cells of the flatworm. Each egg-laying episode consumes all of the precursor-containing vitelline cells. A Dopa-containing protein, vitelline protein 1 (Vp1), has been isolated from B. candida and resembles the primary eggshell layer in its composition. Vp1 has an apparent molecular weight of 34.4 and an acidic pI. Dopa-containing proteins are presumed to be directly involved in the cross-linking reactions that accompany quinone-tanning. The secondary outer layer of the eggshell may serve to adhere the eggshell to the gill. Its amino acid composition is unlike that of the primary eggshell and nothing is known about its precursors.

Amino Acids↗
PubMed1993

Solid-state NMR analysis of crosslinking in mussel protein glue.

Solid-state 13C and 15N NMR spectra have been obtained of intact adhesive plaques from the mussel Geukensia demissa labeled by L-[6-13C,6-15N]lysine. The plaques are rich in a polyphenolic protein glue which has 50 or more repeats of a nonapeptide sequence with one lysine per repeat. The average isotopic 15N enrichment of lysyl epsilon nitrogens in the plaques was 4%. These lysyl amines are not involved in ionic complexes and do not form observable concentrations of covalent crosslinks.

Animals↗