Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences.

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Interspecific Analysis of Sea Urchin Adhesive Composition Emphasizes Variability of Glycans Conjugated With Putative Adhesive Proteins

Frontiers in Marine Science ◽

10.3389/fmars.2021.737886 ◽

2021 ◽

Vol 8 ◽

Author(s):

Lisa Gaspar ◽

Patrick Flammang ◽

Ricardo José ◽

Ricardo Luis ◽

Patrício Ramalhosa ◽

...

Keyword(s):

Sea Urchin ◽

Sea Urchins ◽

Secretory Granules ◽

Paracentrotus Lividus ◽

Single Species ◽

Lectin Histochemistry ◽

Additional Species ◽

Adhesive Organs ◽

Adhesive Proteins ◽

Tube Feet

Sea urchins possess specialized adhesive organs, tube feet. Although initially believed to function as suckers, it is currently accepted that they rely on adhesive and de-adhesive secretions to attach and detach repeatedly from the substrate. Given the biotechnological potential of their strong reversible adhesive, sea urchins are under investigation to identify the protein and glycan molecules responsible for its surface coupling, cohesion and polymerization properties. However, this characterization has only focused on a single species, Paracentrotus lividus. To provide a broader insight into sea urchins adhesion, a comparative study was performed using four species belonging to different taxa and habitats: Diadema africanum, Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. Their tube feet external morphology and histology was studied, together with the ultrastructure of their adhesive secretory granules. In addition, one antibody and five lectins were used on tube foot histological sections and extracts, and on adhesive footprints to detect the presence of adhesion-related (glyco)proteins like those present in P. lividus in other species. Results confirmed that the antibody raised against P. lividus Nectin labels the adhesive organs and footprints in all species. This result was further confirmed by a bioinformatic analysis of Nectin-like sequences in ten additional species, increasing the comparison to seven families and three orders. The five tested lectins (GSL II, WGA, STL, LEL, and SBA) demonstrated that there is high interspecific variability of the glycans involved in sea urchin adhesion. However, there seems to be more conservation among taxonomically closer species, like P. lividus and S. granularis. In these species, lectin histochemistry and lectin blots indicated the presence of high molecular weight putative adhesive glycoproteins bearing N-acetylglucosamine residues in the form of chitobiose in the adhesive epidermis and footprints. Our results emphasize a high selective pressure for conservation of functional domains in large putative cohesive proteins and highlight the importance of glycosylation in sea urchin adhesion with indications of taxonomy-related conservation of the conjugated glycans.

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Cloning and Expression Analysis of Alpha-Tubulin Genes in Water Foxtail (Alopecurus aequalis)

Weed Science ◽

10.1614/ws-d-09-00034.1 ◽

2010 ◽

Vol 58 (2) ◽

pp. 89-95 ◽

Cited By ~ 7

Author(s):

Saima Hashim ◽

Mayumi Hachinohe ◽

Hiroshi Matsumoto

Keyword(s):

Gene Families ◽

Amino Acid Sequences ◽

Cloning And Expression ◽

Dependent Manner ◽

Degenerate Primers ◽

Specific Expression ◽

Alpha Tubulin ◽

Tubulin Genes ◽

Complete Sequences

Tubulins are encoded by small gene families in plants. Here we report the cloning and characterization of water foxtail α-tubulin genes (AaTUA), an economically important weed. The genome of water foxtail contains TUA family consisting of at least four genes. Using degenerate primers, four partial TUA genes were isolated from the complementary DNA (cDNA) of water foxtail. Using the partial gene sequences, specific primers for each TUA gene were designed and full-length TUA cDNAs were isolated. These genes were designated as AaTUA1 to AaTUA4. The deduced amino acid sequences of AaTUA genes showed significant homology to the TUA genes of barley, corn, and Arabidopsis. The coding sequences of the AaTUA1 and AaTUA3 genes were interrupted by three introns and there were four introns in the coding regions of AaTUA2 and AaTUA4. The organ-specific expression of AaTUA genes showed that AaTUA1 and AaTUA4 were predominantly expressed in all organs examined (root, stem, young leaf, mature leaf, and panicle), whereas AaTUA2 was expressed mainly in roots and AaTUA3 was expressed in stem, root, and panicle. Abscisic acid (ABA) and gibberellic acid (GA) differentially induced the expression of AaTUA1 and AaTUA3. Moreover, trifluralin, propham, and caffeic acid induced the expression of all AaTUA genes in a dose-dependent manner except AaTUA2. This is the first report of complete sequences of AaTUA genes and the first characterization of these genes for any Alopecurus species in the literature.

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The Art of Mast Cell Adhesion

Cells ◽

10.3390/cells9122664 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2664

Author(s):

Joanna Pastwińska ◽

Paulina Żelechowska ◽

Aurelia Walczak-Drzewiecka ◽

Ewa Brzezińska-Błaszczyk ◽

Jarosław Dastych

Keyword(s):

Cell Adhesion ◽

Current Knowledge ◽

Living Organism ◽

Cellular Mechanisms ◽

Cellular Processes ◽

Adhesion Sites ◽

Inflammatory Processes ◽

Adhesive Molecules ◽

Adhesive Proteins ◽

Insight Into

Cell adhesion is one of the basic phenomena occurring in a living organism, affecting many other processes such as proliferation, differentiation, migration, or cell viability. Mast cells (MCs) are important elements involved in defending the host against various pathogens and regulating inflammatory processes. Due to numerous mediators, they are contributing to the modulation of many basic cellular processes in a variety of cells, including the expression and functioning of different adhesive molecules. They also express themselves many adhesive proteins, including ICAM-1, ICAM-3, VCAM-1, integrins, L-selectin, E-cadherin, and N-cadherin. These molecules enable MCs to interact with other cells and components of the extracellular matrix (ECM), creating structures such as adherens junctions and focal adhesion sites, and triggering a signaling cascade. A thorough understanding of these cellular mechanisms can create a better understanding of MC biology and reveal new goals for MC targeted therapy. This review will focus on the current knowledge of adhesion mechanisms with the involvement of MCs. It also provides insight into the influence of MCs or MC-derived mediators on the adhesion molecule expression in different cells.

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Temporary adhesion of the proseriate flatworm Minona ileanae

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2019.0194 ◽

2019 ◽

Vol 374 (1784) ◽

pp. 20190194 ◽

Cited By ~ 5

Author(s):

Robert Pjeta ◽

Julia Wunderer ◽

Philip Bertemes ◽

Teresa Hofer ◽

Willi Salvenmoser ◽

...

Keyword(s):

Cost Effective ◽

Adhesive System ◽

Ventral Side ◽

Adhesive Properties ◽

Expression Screening ◽

Antibody Staining ◽

A Genome ◽

Tail Plate ◽

Adhesive Organs ◽

Adhesive Proteins

Flatworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae . We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae . We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue ‘Transdisciplinary approaches to the study of adhesion and adhesives in biological systems’.

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Ribosome Structural Organization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051670 ◽

1974 ◽

Vol 32 ◽

pp. 332-333

Author(s):

James A. Lake

Keyword(s):

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Small Subunit ◽

Structural Studies ◽

X Ray Diffraction ◽

Specific Antibodies ◽

X Ray ◽

E Coli ◽

Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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Conformational Changes in Adhesive Proteins Modulate Their Adhesive Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642686 ◽

1995 ◽

Vol 74 (01) ◽

pp. 253-257 ◽

Cited By ~ 22

Author(s):

Tatiana Ugarova ◽

Francisca R Agbanyo ◽

Edward F Plow

Keyword(s):

Conformational Changes ◽

Adhesive Proteins

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Clues for Understanding the Structure and Function of a Prototypic Human Integrin: The Platelet Glycoprotein IIb/IIIa Complex

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648803 ◽

1994 ◽

Vol 72 (01) ◽

pp. 001-015 ◽

Cited By ~ 124

Author(s):

Juan J Calvete

Keyword(s):

Structure Function ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Primary Role ◽

Clot Retraction ◽

Inhibitory Peptide ◽

Rgd Sequence ◽

Platelet Receptor ◽

Adhesive Proteins ◽

And Function

SummaryThe glycoprotein (GP) IIb/IIIa, a Ca2+-dependent heterodimer, is the major integrin on the platelet plasma membrane. On resting platelets GPIIb/IIIa is maintained in an inactive conformation and serves as a low affinity adhesion receptor for surface-coated fibrinogen, whereas upon platelet activation signals within the cytoplasma alter the receptor function of GPIIb/IIIa (inside-out signalling), which undergoes a measurable conformational change within its exoplasmic domains, and becomes a competent receptor for soluble fibrinogen and some other RGD sequence-containing plasma adhesive proteins. Upon ligand binding, further structural alterations trigger the association of receptor-occupied GPIIb/IIIa complexes with themselves within the plane of the membrane. The simultaneous binding of dimeric fibrinogen molecules to GPIIb/IIIa clusters on adjacent platelets leads to platelet aggregation, which promotes attachment of fibrinogen-GPIIb/IIIa clusters to the cytoskeleton (outside-in signalling). This, in turn, provides the necessary physical link for clot retraction to occur, and generates a cascade of intracellular biochemical reactions which result in the formation of a multiprotein signalling complex at the cytoplasmic domains of GPIIb/IIIa. Glycoprotein IMIIa, also called αIIbβ3 in the integrin nomenclature, plays thus a primary role in both platelet adhesion and thrombus formation at the site of vascular injury. In addition, the human glycoprotein Ilb/IIIa complex is the most thoroughly studied integrin receptor, its molecular biology and major features of its primary structure having been elucidated mainly during the last six years. Furthermore, localization of functionally relevant monoclonal antibody epitopes, determination of the cross-linking sites of inhibitory peptide ligands, proteolytic dissection of the isolated integrin, and analysis of natural and artificial GPIIb/IIIa mutants have recently provided a wealth of information regarding structure-function relationships of human GPIIb/IIIa. The aim of this review is to summarize these many structural and functional data in the perspective of an emerging model. Although most of the interpretations based on structural elements of this initial biochemical model require independent confirmation, they may help us to understand the structure-function relationship of this major platelet receptor, and of other members of the integrin superfamily, as well as to perform further investigations in order to test current hypotheses.

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Complete sequences of 4 viral hemorrhagic septicemia virus IVb isolates and their virulence in northern pike fry

Diseases of Aquatic Organisms ◽

10.3354/dao03171 ◽

2017 ◽

Vol 126 (3) ◽

pp. 211-227 ◽

Cited By ~ 7

Author(s):

RG Getchell ◽

ER Cornwell ◽

S Bogdanowicz ◽

J Andrés ◽

WN Batts ◽

...

Keyword(s):

Northern Pike ◽

Viral Hemorrhagic Septicemia Virus ◽

Viral Hemorrhagic Septicemia ◽

Complete Sequences

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Fabrication of Nanoscale Hydrophobic Regions on Anodic Alumina for Selective Adhesion of Biologic Molecules

MRS Proceedings ◽

10.1557/proc-773-n6.3 ◽

2003 ◽

Vol 773 ◽

Author(s):

Xiefan Lin ◽

Anthony S. W. Ham ◽

Natalie A. Villani ◽

Whye-Kei Lye ◽

Qiyu Huang ◽

...

Keyword(s):

Anodic Alumina ◽

Biological Molecules ◽

Spatial Density ◽

Length Scale ◽

Adhesion Sites ◽

A Cell ◽

Adhesive Molecules ◽

Receptor Clusters ◽

Molecular Bonding ◽

Fabrication Parameters

AbstractStudies of selective adhesion of biological molecules provide a path for understanding fundamental cellular properties. A useful technique is to use patterned substrates, where the pattern of interest has the same length scale as the molecular bonding sites of a cell, in the tens of nanometer range. We employ electrochemical methods to grow anodic alumina, which has a naturally ordered pore structure (interpore spacing of 40 to 400 nm) controlled by the anodization potential. We have also developed methods to selectively fill the alumina pores with materials with contrasting properties. Gold, for example, is electrochemically plated into the pores, and the excess material is removed by backsputter etching. The result is a patterned surface with closely separated islands of Au, surrounded by hydrophilic alumina. The pore spacing, which is determined by fabrication parameters, is hypothesized to have a direct effect on the spatial density of adhesion sites. By attaching adhesive molecules to the Au islands, we are able to observe and study cell rolling and adhesion phenomena. Through the measurements it is possible to estimate the length scale of receptor clusters on the cell surface. This information is useful in understanding mechanisms of leukocytes adhesion to endothelial cells as well as the effect of adhesion molecules adaptation on transmission of extracellular forces. The method also has applications in tissue engineering, drug and gene delivery, cell signaling and biocompatibility design.

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