“Substrate Inhibition” Is One of the Aspects of Substrate Specificity in Vertebrate and Invertebrate Cholinesterases

An analytical review is performed of the literature data on the hydrolysis rate, affinity of substrate to active center, and constants of the substrate inhibition (K _ss) at hydrolysis of the choline (acetyl-, propyonyl-, butyrylcholine, acetyl--methylcholine) and/or of corresponding thiocholine substrates by 59 cholinesterases from 49 different animals (chordate, insects, molluscs, nematodes). The characteristic peculiarities of enzymes from different groups of animals are revealed. The absence of regular changes of parameters of the cholinesterase substrate specificity in the course of evolutionary development is shown.     

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin ��V��3

Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin α_Vβ₃ antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin α_Vβ₃ as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion.Cellular interactions with extracellular matrix proteins are vital for cell survival and tissue maintenance. The attachment of cells to their extracellular matrix (ECM)³ is often mediated by cell surface integrins. As such, integrins are involved in many biological functions such cell migration and proliferation, tissue organization, wound repair, development, and host immune responses. In addition to roles under normal physiological conditions, integrins are involved in the pathogenesis of diseases such as arthritis, cardiovascular disease, inflammation, microbial and parasitic infection, and cancer. Integrins are a family of heterodimeric transmembrane receptors containing one α subunit and one β subunit (1). Often integrins bind to ECM proteins via short RGD motifs within the matrix protein (2). In addition to an RGD motif, fibronectin also contains an upstream PHSRN synergy sequence, which is required for full integrin binding activity (3).Elastin confers elasticity on all vertebrate elastic tissues including arteries, lung, skin, vocal fold, and elastic cartilage (4). Elastin comprises ∼90% of the elastic fiber and is intermingled with fibrillin-rich microfibrils (5). There is a single human tropoelastin gene in which alternative splicing can result in the loss of domains 22, 23, 24, 26A, 30, 32, and 33 (4). Elastin is made from the secreted monomer tropoelastin, which is a 60–72-kDa protein containing repeating hydrophobic and cross-linking domains. Hydrophobic domains are rich in GVGVP, GGVP, and GVGVAP repeats, which can associate by coacervation (6). This association results in structural changes and increased α-helical content (7). The cross-linking domains are lysine-rich. Occasionally these residues are modified to allysine through the activity of members of the family of lysyl oxidase (LOX) and four LOX-like enzymes. During coacervation the allysine and other allysines or specific lysine side chains come into close proximity, allowing nonenzymatic condensation reactions to occur, forming desmosine or isodesmosine cross-links (4). This process gives a highly stable cross-linked elastin matrix which has a half-life of ∼70 years. Members of the serine, aspartate, cysteine, and matrix metalloproteinase families of proteases can degrade elastin (8). The resulting elastin peptides have effects on ECM synthesis and cell attachment, migration, and proliferation (9).The consequences of mutated or hemizygous elastin in the hereditary, connective tissue disorders cutis laxa, supravalvular aortic stenosis, and Williams-Beuren syndrome highlight the elastins essential role in elastic tissue function (10). Elastin is the major protein in large elastic blood vessels such as the aorta, where it is likely to inhibit the proliferation of vascular smooth muscle cells and so preventing vessel occlusion (11), which is a major cause of death in developed countries. Previous studies have shown that human and bovine tropoelastin can bind directly to a variety of cell types directly through a number of cell surface receptors (12–14) and also bind indirectly to cells through ECM proteins such as fibulin-5 (15, 16).A mechanism by which elastin binds to cells is via the 67-kDa elastin-binding protein (EBP), which is a peripheral membrane splice variant of β-galactosidase. The EBP forms a complex with the integral membrane proteins carboxypeptidase A and sialidase, forming a transmembrane elastin receptor (12). The binding site for the EBP has been mapped to the consensus sequence XGXXPG within elastin and in particular to VGVAPG within exon 24 (17). The binding of elastin to the EBP results in cell morphological changes (18, 19), chemotaxis (20), decreased cell proliferation (21), and angiogenesis (22). Knockouts of β-galactosidase, which remove the EBP, display correctly deposited elastin (27). Additionally tropoelastin actively promotes cell adhesion, whereas VGVAPG does not. These observations imply that receptors other than EBP can interact with elastin.Other studies have proposed a second mechanism involving the necessity of cell surface heparan and chondroitin sulfate-containing glycosaminoglycans for bovine chondrocyte interaction with bovine tropoelastin (14). Peptide binding analysis implicated the last 17 amino acids at the C terminus of bovine tropoelastin in this cell adhesive activity, with higher binding requiring the C-terminal 25 amino acids. This region is of interest, as in humans a mutation of Gly-773 to Asp in exon 33 results in blocked elastin network assembly and modulates cell binding to a peptide corresponding to exons 33 and 36 of human tropoelastin (28). Indeed Broekelmann et al. (14) have shown that synthetic peptides containing the C-terminal 29 amino acids of bovine tropoelastin possess cell adhesive activity; however, when the G773D mutation was incorporated into the peptide, it prevented cell adhesion to that peptide.Although tropoelastin does not contain an RGD motif, other data identified a third mechanism involving direct interaction between integrin α_vβ₃ and human tropoelastin (13, 29). This interaction was also localized to the C-terminal domains of tropoelastin.More recent data has shown that human umbilical vein endothelial cells can adhere to recombinant fragments of human tropoelastin (30, 31). In contrast to other data, regions encoded by the N-terminal exons (1–18), the central exons (18–27), and the C-terminal exons (18–36) all supported human umbilical vein endothelial cell attachment.Although a previous study has shown a direct interaction between purified integrin α_vβ₃ and human tropoelastin (13), the integrin dependence of cell adhesion to tropoelastin had not been demonstrated. Here we demonstrate that human dermal fibroblasts adhere to recombinant human tropoelastin and that inhibitors of the elastin-binding protein and cell surface heparan sulfate have no effect on cell adhesion. In contrast, cell adhesion was dependent upon the presence of divalent cations, indicating integrin dependence. Inhibitory monoclonal antibodies identified integrin α_Vβ₃ as the major receptor necessary for fibroblast adherence and spreading onto human tropoelastin. The binding motif for integrin-mediated cell adhesion is unknown; therefore, through the use of synthetic peptides, the adhesive activity was localized to the extreme C-terminal GRKRK motif of tropoelastin. This data present a novel mechanism for cell adhesion to human tropoelastin and identify a novel integrin binding motif within tropoelastin.     

IGF-I Receptor, Cell–Cell Adhesion, Tumour Development and Progression

The insulin-like growth factor I receptor (IGF-IR) has been implicated in the development and progression of many common cancers and other neoplastic diseases. The tumorigenic potential of IGF-IR relies on its antiapoptotic and transforming activities. The molecular mechanisms by which IGF-IR controls the proliferation and survival of tumour cells have been extensively studied and many pathways have been delineated. However, the role of IGF-IR in the regulation of non-mitogenic cell functions is less well understood. Here we focus on IGF-IR-dependent cell-cell adhesion. Limited studies suggested that IGF-IR can regulate cell aggregation and intercellular adhesion mediated by cadherins and cadherin-associated proteins. We review the mechanisms of this process and discuss the impact of IGF-IR-dependent cell-cell adhesion on the phenotype of tumour cells.     

THE THREE G’s Meeting the Invertebrate Cell Culture Pioneers:Goldschmidt,Gaw,and Grace

It was my good fortune to meet personally the three invertebrate cell culture pioneers,Richard Goldschmidt,Zan-Yin Gaw,and Thomas D.C.Grace (Fig.1).In 1951 I met Goldschmidt at a symposium in Cold Spring Harbor,but I only knew that he was a prominent geneticist.I had no idea about his insect cell culture work at Yale University and daily contacts with Ross G.Harrison.In 1959 Zan Yin Gaw in Wuhan successfully cultured monolayers of silkworm cells     

A Review of: “Cell Adhesion Fundamentals and Biotechnological Applications”

Abstract

M.A. Hjortso and J.W. Roos, Eds. Marcel Dekker, New York, 1994; hardbound pp. xi + 273, $135.00     

Plasticity of the ��-Trefoil Protein Fold in the Recognition and Control of Invertebrate Predators and Parasites by a Fungal Defence System

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.     

The Cell Wall of the Arabidopsis Pollen Tube��Spatial Distribution, Recycling, and Network Formation of Polysaccharides

The pollen tube is a cellular protuberance formed by the pollen grain, or male gametophyte, in flowering plants. Its principal metabolic activity is the synthesis and assembly of cell wall material, which must be precisely coordinated to sustain the characteristic rapid growth rate and to ensure geometrically correct and efficient cellular morphogenesis. Unlike other model species, the cell wall of the Arabidopsis (Arabidopsis thaliana) pollen tube has not been described in detail. We used immunohistochemistry and quantitative image analysis to provide a detailed profile of the spatial distribution of the major cell wall polymers composing the Arabidopsis pollen tube cell wall. Comparison with predictions made by a mechanical model for pollen tube growth revealed the importance of pectin deesterification in determining the cell diameter. Scanning electron microscopy demonstrated that cellulose microfibrils are oriented in near longitudinal orientation in the Arabidopsis pollen tube cell wall, consistent with a linear arrangement of cellulose synthase CESA6 in the plasma membrane. The cellulose label was also found inside cytoplasmic vesicles and might originate from an early activation of cellulose synthases prior to their insertion into the plasma membrane or from recycling of short cellulose polymers by endocytosis. A series of strategic enzymatic treatments also suggests that pectins, cellulose, and callose are highly cross linked to each other.Upon contact with the stigma, the pollen grain swells through water uptake and develops a cellular protrusion, the pollen tube. During its growth in planta, the pollen tube invades the transmitting tissue of the pistil and finds its way to the ovary to deliver the male gametes for double fertilization to happen (Heslop-Harrison, 1987). Depending on the species, pollen tubes can grow extremely rapidly both in planta and in in vitro conditions. To fulfill its biological function, the pollen tube has to (1) adhere to and invade transmitting tissues (Hill and Lord, 1987; Lennon et al., 1998), (2) provide physical protection to the sperm cells, and (3) control its own shape and invasive behavior (Parre and Geitmann, 2005b; Geitmann and Steer, 2006). For all of these functions, the pollen tube cell wall plays an important regulatory and structural role. Although the pollen tube does not form a conventional secondary cell wall layer, its wall is assembled in two phases. The “primary layer” is mainly formed of pectins and other matrix components secreted at the apical end of the cell. The “secondary layer” is assembled by the deposition of callose in more distal regions of the cell (Heslop-Harrison, 1987). Depending on the species, cellulose microfibrils have been found to be associated either with the outer pectic or with the inner callosic layer. Unlike most other plant cells, cellulose is not very abundant representing only 10% of total neutral polysaccharides in Nicotiana alata pollen tubes, whereas callose accounts for more than 80% in this species (Schlüpmann et al., 1994).The biochemical composition of the pollen tube cell wall has been well characterized in many species such as Lilium longiflorum (Lancelle and Hepler, 1992; Jauh and Lord, 1996), tobacco (Nicotiana tabacum; Kroh and Knuiman, 1982; Geitmann et al., 1995; Ferguson et al., 1998; Derksen et al., 2011), Petunia hybrida (Derksen et al., 1999), Pinus sylvestris (Derksen et al., 1999), and Solanum chacoense (Parre and Geitmann, 2005a). But for Arabidopsis (Arabidopsis thaliana), the model for plant molecular biology studies (Arabidopsis Genome Initiative, 2000), there is a striking lack of quantitative information concerning the composition of the pollen tube cell wall as well as the spatial distribution of its components. This is all the more surprising because numerous mutants defective in enzymes involved in cell wall synthesis exhibit a pollen tube phenotype (for example, Jiang et al., 2005; Nishikawa et al., 2005; Wang et al., 2011). Two studies have characterized the Arabidopsis pollen germinating in vitro (Derksen et al., 2002) and in vivo (Lennon and Lord, 2000), but both are qualitative rather than quantitative. A biochemical study by Dardelle and coworkers investigated the cell wall sugar composition in a more quantitative way but does not provide any detailed spatial information (Dardelle et al., 2010; Lehner et al., 2010). This lack of information is not surprising given that until recently Arabidopsis pollen was known to be rather challenging to germinate reproducibly in vitro and more difficult to manipulate than the pollen of many other plant species (Bou Daher et al., 2009). With the publication of optimized methods for in vitro germination (Boavida and McCormick, 2007; Bou Daher et al., 2009), it has become much more feasible to germinate healthy-looking Arabidopsis pollen tubes in vitro in a highly reproducible way.The precisely controlled spatial distribution of biochemical components in the pollen tube cell wall is crucial for shape generation and maintenance of this perfectly cylindrical cell (Geitmann and Parre, 2004; Aouar et al., 2010; Fayant et al., 2010; Geitmann, 2010). The pollen tube, therefore, represents an ideal model system to study the link between intracellular signaling, biochemistry, cell mechanical properties, and morphogenesis in plant cells. Because of its typically fast growth rates, it responds quickly to any environmental triggers such as pharmacological, hormone, or enzymatic treatments. Adding Arabidopsis to the group of commonly studied pollen tube species is particularly timely, because one-third of the approximately 800 cell wall synthesis genes identified in this species are expressed in or are specific to its pollen (Pina et al., 2005). Therefore, the Arabidopsis pollen tube has become a valuable system for cell wall studies, especially with the increasing availability of cell wall mutant lines (Liepman et al., 2010).Here we describe the biochemical composition of the Arabidopsis pollen tube cell wall grown in in vitro conditions using immunocytochemical labeling coupled with epifluorescence and electron microscopic techniques. Rather than relying on imaging alone, we developed a quantitative strategy to assess the precise spatial distribution of cell wall components. This quantitative approach will provide an important tool and baseline dataset for the investigation of mutant phenotypes and for the interpretation of pharmacological studies. Furthermore, we used selective and strategically combined enzymatic digestions to determine the degree of connectivity between the individual types of cell wall polysaccharide networks.     

Cell Adhesion: Separation of p120's Powers?

The catenin p120 is involved in many processes, including cell-cell adhesion and cancer. Recent work explores whether p120 independently regulates two key binding partners, RhoGTPase and cadherin.     

Leaders in Cell Adhesion: An Interview with Richard Hynes,Pioneer of Cell–Matrix Interactions

Abstract

On a recent visit Richard O Hynes, FRS, HHMI, Daniel K. Ludwig Professor for Cancer Research at the Koch Institute for Integrative Cancer Research, MIT, graciously agreed to be interviewed in person for the first in Cell Communication and Adhesion's series on “Leaders in Cell Adhesion”. In this interview we discussed three things: 1) the early role of family, mentors, and luck on his career path; 2) his major discoveries of fibronectin, integrins and the evolution of extracellular matrix proteins; and 3) his role in, and thoughts on, current science policy. This interview reveals his characteristic calmness and infectious optimism, his spontaneous and down to earth sense of humor, and his great ability to place scientific questions in perspective. The interview, carried out on April 30^th 2013 is reported here verbatim with only minor editing for clarity.     

Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function

In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLe^x) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2β1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLe^x and lower α2,6-sialic acid content on the glycans of their α2β1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLe^x and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2β1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion.     

Cell Adhesion: Sorting out Cell Mixing with Echinoid?

Cadherins control intercellular adhesion in epithelial cells. This property relies on the ability to recruit actin filaments at adherens junctions via beta-catenin and alpha-catenin. A recent study shows that Echinoid, a member of the immunoglobulin domain containing protein family, is a modulator of intercellular adhesion in Drosophila that controls cell sorting.