Microciona Prolifera Research Articles

Proteomic analysis of the sponge Aggregation Factor implicates an ancient toolkit for allorecognition and adhesion in animals

The discovery that sponges (Porifera) can fully regenerate from aggregates of dissociated cells launched them as one of the earliest experimental models to study the evolution of cell adhesion and allorecognition in animals. This process depends on an extracellular glycoprotein complex called the Aggregation Factor (AF), which is composed of proteins thought to be unique to sponges. We used quantitative proteomics to identify additional AF components and interacting proteins in the classical model, Clathria prolifera, and compared them to proteins involved in cell interactions in Bilateria. Our results confirm MAFp3/p4 proteins as the primary components of the AF but implicate related proteins with calx-beta and wreath domains as additional components. Using AlphaFold, we unveiled close structural similarities of AF components to protein domains in other animals, previously masked by the mutational decay of sequence similarity. The wreath domain, believed to be unique to the AF, was predicted to contain a central beta-sandwich of the same organization as the vWFD domain (also found in extracellular, gel-forming glycoproteins in other animals). Additionally, many copurified proteins share a conserved C-terminus, containing divergent immunoglobulin (Ig) and Fn3 domains predicted to serve as an AF-interaction interface. One of these proteins, MAF-associated protein 1, resembles Ig superfamily cell adhesion molecules and we hypothesize that it may function to link the AF to the surface of cells. Our results highlight the existence of an ancient toolkit of conserved protein domains regulating cell-cell and cell-extracellular matrix protein interactions in all animals, and likely reflect a common origin of cell adhesion and allorecognition.

Clathriketal, a new tricyclic spiroketal compound from marine sponge Clathria prolifera attenuates serine exopeptidase dipeptidyl peptidase-IV

An undescribed tricyclic spiroketal compound clathriketal was purified from the solvent extract of the Microcionidae sponge Clathria prolifera, and was characterised as 7-(hydroxymethyl)-13-methoxy-3,11-dimethyl-4-oxo-octahydrospiro[chromene-9,13-pyran]-11-yl propionate by spectroscopic experiments. Clathriketal exhibited significant anti-hyperglycemic property by attenuating serine protease dipeptidyl peptidase-IV (IC50 0.37 mM), and its activity was comparable with the standard diprotin A (IC50 0.31 mM). The spiroketal also exhibited significant inhibitory potentials against carbolytic enzymes α-glucosidase (IC50 0.43 mM) and α-amylase (IC50 0.41 mM). Superior antioxidant properties of clathriketal against the oxidants, 2, 2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid and 2, 2-diphenyl-1-picrylhydrazyl (IC50 ∼1.2 mM) also reinforced its promising anti-hyperglycemic activity. Considerably greater topological surface area (91.29) coupled with lesser steric parameters of clathriketal, as elucidated from the structure-activity relationship analyses could further ascribe the improved ligand-receptor interactions resulting in its prospective anti-hyperglycemic activity. Molecular docking analysis of clathriketal with dipeptidyl peptidase-IV recorded lesser binding energy (–9.63 kcal/mol), which further corroborated its prospective antihyperglycemic activity.

Two new pyrrolo-2-aminoimidazoles from a Myanmarese marine sponge, Clathria prolifera.

Marine organisms such as marine sponges and soft corals are valuable sources of pharmacologically active secondary metabolites. In our ongoing research on the discovery of new secondary metabolites from marine organisms, two new pyrrolo-2-aminoimidazoles, clathriroles A (1) and B (2), were isolated from the water-soluble portion prepared from the methanol and acetone (2:1) extract of the marine sponge, Clathria prolifera, collected in Myanmar. The chemical structures of the isolated compounds were determined using extensive spectroscopic techniques, including NMR, HRESIMS, IR, and optical rotation, and comparisons with the reported literature. The spectroscopic analyses of 1 and 2 suggested that 1 is an enantiomer of antifungal N-methylmanzacidin C isolated from the marine sponge Axinella brevistyla, whereas 2 is a diastereomer of manzacidin D at C-11 isolated from the marine sponge Astrosclera willeyana. To the best of our knowledge, this is the first report of the isolation of the pyrrolo-2-aminoimidazole compounds from C. prolifera. Furthermore, in contrast to the potency of N-methylmanzacidin C against Saccharomyces cerevisiae, the antifungal assay revealed that 1 and 2 lack any activity against this strain. Thus, these observations may suggest that the absolute configurations at both C-9 and C-11 play an important role in controlling the antifungal activity of this type of compound.

Identfication of Microbial Symbionts Harbored by the Chesapeake Bay Sponge, Clathria prolifera

Marine sponges are a rich source of pharmaceutically‐interesting compounds. Many of these chemicals are produced by bacterial symbionts that live in tight association with the host sponge. Given the potential therapeutic importance of some of these compounds, there is interest in assessing factors that contribute to our ability to cultivate these bacteria so that compounds may be mass‐produced. We explored carotenoid production in marine sponges because bacterial symbionts likely produce these compounds given that animals are incapable of synthesizing the pigment. Thus, exploring the manner of pigment production in the sponge may shed light on metabolic and biochemical integration between the symbiont and host. We isolated more than unique 50 pigmented colonies cultured from the progeny of the Chesapeake Bay sponge, Clathria prolifera. Pigmented colonies were specifically selected to begin to answer questions about the incorporation of potential antioxidants into the sponge. Freezer stocks were prepared from single colonies and were then grown in marine broth overnight at room temperature. Genomic DNA was isolated from these colonies and nested PCR of the DNA was performed to amplify the 16S rDNA. Bands were cut out from these PCR samples, gel extracted, and sequenced. We created phylogenetic trees to look for relatedness among the microbial species identified. We believe that our study may begin to identify and answer questions about the symbiotic structure harbored by C. prolifera.

Identification of biosynthetic gene clusters from sponge‐associated microbes (618.44)

Greater than 90% of microbes living in soil are unculturable due to the complex nutrient and temperature conditions required for growth. These microorganisms represent a potential untapped source of natural products that may be developed for biotechnological and pharmaceutical uses. Most notably, microorganisms with phosphopantetheinyl transferase (PPTase) activity are of high interest due to the role PPTase has in activating non‐ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene clusters. These gene clusters synthesize complex natural products utilized by the microorganism or its host for selective advantages. We screened for the presence of NRPS and PKS gene clusters in several pigmented microbes previously isolated from a Chesapeake Bay sponge (Clathria prolifera). Genomic DNA was isolated from each of these microbes and we attempted to construct metagenomic libraries. We goal is to screen the libraries for PPTase activity. We extended this study to include microbial DNA isolated from soil samples taken around the University of Richmond campus. The isolated environmental DNA (eDNA) was tested for the presence of NPRS and PKS gene clusters as well. This on‐going project seeks to identify new biosynthetic gene clusters from microbial sources.Grant Funding Source: University of Richmond Arts and Sciences

Model system for cell adhesion mediated by weak carbohydrate-carbohydrate interactions.

The multivalent carbohydrate-carbohydrate interaction between membrane-anchored epitopes derived from the marine sponge Microciona prolifera has been explored by colloidal probe microscopy. An in situ coupling of sulfated and non-sulfated disaccharides to membrane-coated surfaces was employed to mimic native cell-cell contacts.The dynamic strength of the homomeric self-association was measured as a function of calcium ions and loading rate. A deterministic model was used to estimate the basic energy landscape and number of participating bonds in the contact zone.

Weak Carbohydrate-Carbohydrate Interactions Measured by Colloidal Probe Microscopy

The weak calcium mediated interaction between membrane based carbohydrates derived from the marine sponge Microciona prolifera has been investigated by colloidal probe microscopy. An in situ synthesis of glycolipids was employed to measure the dynamic strength of homomeric self-association between sulfated and non-sulfated oligosaccharides. For this purpose an in situ protocol has been developed to entirely rely on a defined synthetic system with adjustable composition and physical properties. Since thermal undulation of membranes renders analysis of binding affinity cumbersome we rely on solid supported membranes with preserved fluidity and minimal non-specific interaction. We investigate the attraction of carbohydrates derived from Microciona prolifera with a focus on the role of calcium ions, the necessity of the sulfate group, membrane fluidity, and loading rate. Membrane fluidity is of particular interest considering that lateral organization of ligand-receptors into small-scale clusters as a result of competition between binding enthalpy and mixing entropy is only possible in laterally mobile matrices.We found that nanoclusters consisting of 4-8 homomeric bonds were formed in the contact area between colloidal probe and solid supported membrane. Depletion of calcium or removal of the sulfato group resulted both in a disappearance of specific interactions.

Recreational boats as potential vectors of marine organisms at an invasion hotspot

With more than 200 aquatic nonindigenous species (NIS), San Francisco Bay (California, USA) is among the world's most invaded harbors. Hard-substratum benthic (biofouling) organisms, which dominate NIS richness, have arrived primarily as a result of shipping and aquaculture activity over past centuries. To date there has been no assessment of the leisure craft vector in the Bay. We aimed to characterize (1) biofouling on boats' submerged surfaces and (2) boater behavior likely to affect the risk of NIS transfers. We used an underwater pole-cam, specimen collections, and a boater questionnaire to quantify the extent and composition of biofouling on recreational boats and to eval- uate boater behavior at a subset of the Bay's marinas. Several NIS, already established within the Bay, were recorded from vessel hulls, including the bryozoans Bugula neritina, Membranipora chesa- peakensis and Watersipora sp., the ascidians Botrylloides violaceus, Styela clava and Ciona intesti- nalis, the polychaete Ficopomatus enigmaticus, and the sponge Clathria prolifera. Only 16% of ques- tionnaire respondents had traveled to sites outside the Bay in the previous 12 mo. Frequency of hull painting and cleaning varied substantially, but we did not find strong patterns of biofouling extent associated with hull husbandry or boat usage. The potential for within-Bay and coastwise regional spread of NIS is high, and recreational boats probably interact in close proximity to other vectors (e.g. commercial ships), causing a ratchet effect of vector events; however, there remains a gap in under- standing the levels and condition of biofouling on transient boats. Transient vessels from San Fran- cisco Bay and other West Coast sites should be the focus of future studies to evaluate the extent to which organisms are being transferred among bays and how vector management could be applied to prevent NIS transfers and impacts.

Comparison of the Bacterial Communities of Wild and Captive Sponge Clathria prolifera from the Chesapeake Bay

The red-beard sponge Clathria prolifera, which is widely distributed in the USA, has been widely used as a model system in cell biology and has been proposed as a suitable teaching tool on biology and environmental sciences. We undertook the first detailed microbiological study of this sponge on samples collected from the Chesapeake Bay. A combination of culture-based studies, denaturing gradient gel electrophoresis, and bacterial community characterization based on 16S rRNA gene sequencing revealed that C. prolifera contains a diverse assemblage of bacteria that is different from that in the surrounding water. C. prolifera individuals were successfully maintained in a flow-through or recirculation aquaculture system for over 6 months and shifts in the bacterial assemblages of sponges in aquaculture compared with wild sponges were examined. The proteobacteria, bacteroidetes, actinobacteria, and cyanobacteria represented over 90% of the species diversity present in the total bacterial community of the wild C. prolifera. Actinobacteria, cyanobacteria, and spirochetes were not represented in clones obtained from C. prolifera maintained in the aquaculture system although these three groups comprised ca. 20% of the clones from wild C. prolifera, showing a significant effect of aquaculture on the bacterial community composition. This is the first systematic characterization of the bacterial community from a sponge found in the Chesapeake Bay. Changes in sponge bacterial composition were observed in sponges maintained in aquaculture and demonstrate the importance of monitoring microbial communities when cultivating sponges in aquaculture systems.

Cover Picture: Assessing Carbohydrate–Carbohydrate Interactions by NMR Spectroscopy: The Trisaccharide Epitope from the Marine Sponge Microciona prolifera (ChemBioChem 3/2009)

AbstractThe cover picture shows a 3D model, derived from molecular dynamics simulations, of the trisaccharides that result from the mutual interaction in solution of the synthetically prepared trisaccharide epitope of the marine sponge Microciona prolifera and gold glyconanoparticles, displaying the same sugar moiety, in the presence of calcium. As can be seen, the trisaccharides are gathered together by bridged calcium atoms, thus precluding the dissociation of the complex. The different trisaccharide entities interact in a parallel fashion and are layered in pairs. Thus, they show a segmental type of interaction that could explain the actual interaction in Nature of the native g‐200 acidic glycan present in the MAFp3 glycoprotein, with the polysaccharide–polysaccharide interaction taking place via the trisaccharide epitopes. For further details, see the article by J. Kamerling, J. Jiménez‐Barbero et al. on p. 511 ff. The picture of Microciona prolifera was taken from http://www.uniprot.org/taxonomy/27928magnified image

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge Microciona prolifera

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca(2+)-independent cell-binding domain and (ii) a Ca(2+)-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide beta-D: -GlcpNAc3S-(1-->3)-alpha-L: -Fucp and a pyruvated trisaccharide beta-D: -Galp4,6(R)Pyr-(1-->4)-beta-D: -GlcpNAc-(1-->3)-alpha-L: -Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca(2+)-ions. To determine binding forces of the carbohydrate-carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca(2+)-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing beta-D: -GlcpNAc-(1-->3)-alpha-L: -Fucp-(1-->O)(CH(2))(3)S(CH(2))(6)S- was found to be quantized in integer multiples of 30 +/- 6 pN. No binding was observed between the two monolayers in the absence of Ca(2+)-ions. Cd(2+)-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing beta-D: -Galp4,6(R)Pyr-(1-->4)-beta-D: -GlcpNAc-(1-->3)-alpha-L: -Fucp-(1-->O)(CH(2))(3)S(CH(2))(6)S- did not show a binding in the presence of Ca(2+)-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca(2+)-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.

Gold nanoparticles coated with a pyruvated trisaccharide epitope of the extracellular proteoglycan of Microciona prolifera as potential tools to explore carbohydrate-mediated cell recognition

The species-specific cell adhesion in the marine sponge Microciona prolifera involves the interaction of an extracellular proteoglycan-like macromolecular complex, otherwise known as aggregation factor. In the interaction, two highly polyvalent functional domains play a role: a cell-binding and a self-interaction domain. The self-recognition has been characterized as a Ca(2+)-dependent carbohydrate-carbohydrate interaction of repetitive low affinity carbohydrate epitopes. One of the involved epitopes is the pyruvated trisaccharide beta-d-Galp4,6(R)Pyr-(1-->4)-beta-d-GlcpNAc-(1-->3)-l-Fucp. To evaluate the role of this trisaccharide in the proteoglycan-proteoglycan self-recognition, beta-d-Galp4,6(R)Pyr-(1-->4)-beta-d-GlcpNAc-(1-->3)-alpha-l-Fucp-(1-->O)(CH(2))(3)S(CH(2))(6)SH was synthesized, and partially converted into gold glyconanoparticles. These mimics are being used to explore the self-interaction phenomenon for the trisaccharide epitope, via TEM aggregation experiments (gold glyconanoparticles) and atomic force microscopy (AFM) experiments (self assembled monolayers; binding forces).

The Early Evolution of the Phosphagen Kinases—Insights from Choanoflagellate and Poriferan Arginine Kinases

Arginine kinase (AK) is a member of a large family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases. AKs are present in certain protozoans, sponges, cnidarians, and both lophotrochozoan and ecdysozoan protostomes. Another phosphagen kinase, creatine kinase (CK), is found in sponges, cnidarians, and both deuterostome and protostome groups but does not appear to be present in protozoans. To probe the early evolution of phosphagen kinases, we have amplified the cDNAs for AKs from three choanoflagellates and from the hexactinellid sponge Aphrocallistes beatrix and the demosponges Suberites fuscus and Microciona prolifera. Phylogenetic analysis using maximum likelihood of these choanoflagellate and sponge AKs with other AK sequences revealed that the AK from the choanoflagellate Monosiga brevicollis clusters with the AK from the glass sponge Aphrocallistes and is part of a larger cluster containing AKs from the demosponges Suberites and Microciona as well as basal and protostome invertebrates. In contrast, AKs from Codonosiga gracilis and Monosiga ovata form a distinct cluster apart from all other AK sequences. tBLASTn searches of the recently released M. brevicollis genome database showed that this species has three unique AK genes-one virtually identical to the M. brevicollis cDNA and the other two showing great similarity to C. gracilis and M. ovata AKs. Three distinct AK genes are likely present in choanoflagellates. Two of these AKs display extensive similarity to both CKs and an AK from sponges. Previous work has shown CK evolved from an AK-like ancestor prior to the divergence of sponges. The present results provide evidence suggesting that the initial gene duplication event(s) leading to the CK lineage may have occurred before the divergence of the choanoflagellate and animal lineages.

Cyclosporin A Suspends Transplantation Reactions in the Marine SpongeMicrociona prolifera

Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.

Gold Glyconanoparticles as Probes to Explore the Carbohydrate‐Mediated Self‐Recognition of Marine Sponge Cells

Cell aggregation in the red-beard marine sponge Microciona prolifera is mediated by a 2 10 kDa proteoglycan-like macromolecular aggregation factor (MAF), and is based on two highly polyvalent functional properties; a Ca-dependent proteoglycan self-interaction and a Ca-independent cell-binding activity. MAF, the first circular proteoglycan described, is composed of two N-glycosylated proteins, MAFp3 and MAFp4, with twenty units of each glycoprotein forming the central ring and the radiating arms, respectively. Each MAFp3 carries one or two copies of a 200 kDa acidic glycan, g-200, whereas each MAFp4 carries about 50 copies of a 6 kDa glycan, g-6. The MAFp4 arms of the sunburst-like proteoglycan are linked to cell-surface binding receptors, while the MAFp3 ring exposes the g-200 glycans so that they can engage in the Ca-dependent self-association (for a detailed review, see ref. [4]). By making use of MAF-specific monoclonal antibodies, it could be demonstrated that the self-association of MAF occurs through highly repetitive epitopes on the g-200 glycan. One of these epitopes was shown to be the sulfated disaccharide GlcpNAc3S(b1–3)Fucp. To gain insight into the role of carbohydrate interactions in MAF self-aggregation, we designed a challenging system for mimicking the g-200 self-association. By using the synthetic sulfated disaccharide, multivalently presented as a bovine serum albumin conjugate, and surface plasmon resonance spectroscopy, it was shown that Ca-dependent carbohydrate self-recognition is a major force in the g-200 association phenomenon. Gold glyconanoparticles have been successfully used as inert multivalent systems to explore either carbohydrate self-interactions or carbohydrate binding to proteins. In the present study, water-soluble gold glyconanoparticles coated with synthetic carbohydrates related to the sulfated disaccharide fragment (Scheme 1) were used as multivalent systems to investigate the g-200 glycan–glycan interaction by transmission electron microscopy (TEM). Very recently, an NMR study of intact MAF glycans suggested the presence of a-Fuc residues. However, earlier structural analysis of oligosaccharide fragments obtained from a partial acid hydrolysate of the g-200 glycan could not identify the anomeric configuration of the fucose residue in these fragments. 16] Therefore, gold glyconanoparticles coated with the aor the b-anomer (Au-1a and Au-1b) of the native sulfated disaccharide epitope were used in the aggregation experiments. The importance of each of the two monosaccharide units for the self-recognition process of the disaccharide epitope was determined by studying the gold glyconanoparticles Au-2 and Au-3. The three gold glyconanoparticle systems Au-4 (a-l-Fucp replaced by a-l-Galp), Au-5 (bd-GlcpNAc3S replaced by b-d-GlcpNAc), and Au-6 (b-dGlcpNAc3S replaced by b-d-Glcp3S) were used to evaluate the relevance of the modified sites in the self-recognition process. Scheme 1. Gold glyconanoparticles Au-1a/b to Au-6, related to the MAF sulfated disaccharide epitope.

Anti-predator defenses in western North Atlantic sponges with evidence of enhanced defense through interactions between spicules and chemicals

We examined anti-predator defenses in 4 species of temperate sponge from Long Island Sound using a common hermit crab (Pagurus longicarpus) as predator in pair-wise feeding prefer- ence tests comparing palatable control versus treatment food. Only Haliclona loosanoffi failed to deter hermit crab feeding. The other 3 species reduced P. longicarpus feeding rates, although the mechanism of deterrence differed for each sponge. Spicules and crude extracts interacted to enhance deterrence in experiments involving Microciona prolifera because combining spicules and crude extract in artificial food significantly reduced crab feeding rates by 53%. When added to artificial food in isolation, neither spicules nor crude extract from M. prolifera significantly altered feeding rates of the crabs. Crude extracts, spicules and a combination of spicules and crude extract isolated from Halichondria bowerbanki all significantly reduced crab feeding rates compared to consumption of control food. Structural materials were significantly deterrent in feeding trials involving Cliona celata (32% reduction in feeding), but artificial food containing either crude extract or crude extract plus spicules was significantly preferred over control food. Both H. bowerbanki and C. celata pro- duce spicules that average ≥285 µm in length, which is above the threshold length that has been hypothesized to deter predators. The data involving M. prolifera, H. bowerbanki, and C. celata indi- cate that temperate sponges use chemical and structural defenses against potential predators, and that structural and chemical defenses interact in M. prolifera. Results of this and other studies indicate that it may be fruitful to look for additive and synergistic defenses in other sponge species, especially those found to be chemically undefended when compounds were tested in isolation.

Synthesis of Gold Glyconanoparticles: Possible Probes for the Exploration of Carbohydrate‐Mediated Self‐Recognition of Marine Sponge Cells.

The first step in marine sponge cell recognition and adhesion operates via a calcium-dependent proteoglycan− proteoglycan interaction. For the marine sponge Microciona prolifera, one of the carbohydrate epitopes involved in the proteoglycan self-recognition is a sulfated disaccharide [GlcpNAc3S(β1−3)Fucp]. Earlier surface plasmon resonance studies have demonstrated that the proteoglycan self-recognition can be mimicked with synthetic β-D-GlcpNAc-(13)-α-L-Fucp-(1O), when multivalently presented by conjugation with bovine serum albumin. Here, the straightforward synthesis of water-soluble gold glyconanoparticles coated with the glycosides β-D-GlcpNAc3S-(13)-α-L-Fucp-(1O)(CH2)3S(CH2)6SH, β-D-GlcpNAc3S-(13)-β-L-Fucp-(1O)(CH2)3S(CH2)6SH, β-D-GlcpNAc3S-(1O)(CH2)3S(CH2)6SH, α-L-Fucp-(1O)(CH2)3S(CH2)6SH, β-D-Glcp-NAc3S-(13)-α-L-Galp-(1O)(CH2)3S(CH2)6SH, β-D-Glcp-NAc-(13)-α-L-Fucp-(1O)(CH2)3S(CH2)6SH, and β-D-Glcp3S-(13)-α-L-Fucp-(1O)(CH2)3S(CH2)6SH is presented. Such supramolecular structures are excellent probes for studying carbohydrate−carbohydrate interactions by transmission electron microscopy, thereby generating information on the molecular level about the role of different functionalities in the self-recognition process. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Synthesis of Gold Glyconanoparticles: Possible Probes for the Exploration of Carbohydrate‐Mediated Self‐Recognition of Marine Sponge Cells

AbstractThe first step in marine sponge cell recognition and adhesion operates via a calcium‐dependent proteoglycan− proteoglycan interaction. For the marine sponge Microciona prolifera, one of the carbohydrate epitopes involved in the proteoglycan self‐recognition is a sulfated disaccharide [GlcpNAc3S(β1−3)Fucp]. Earlier surface plasmon resonance studies have demonstrated that the proteoglycan self‐recognition can be mimicked with synthetic β‐D‐GlcpNAc‐(1→3)‐α‐L‐Fucp‐(1→O), when multivalently presented by conjugation with bovine serum albumin. Here, the straightforward synthesis of water‐soluble gold glyconanoparticles coated with the glycosides β‐D‐GlcpNAc3S‐(1→3)‐α‐L‐Fucp‐(1→O)(CH2)3S(CH2)6SH, β‐D‐GlcpNAc3S‐(1→3)‐β‐L‐Fucp‐(1→O)(CH2)3S(CH2)6SH, β‐D‐GlcpNAc3S‐(1→O)(CH2)3S(CH2)6SH, α‐L‐Fucp‐(1→O)(CH2)3S(CH2)6SH, β‐D‐Glcp‐NAc3S‐(1→3)‐α‐L‐Galp‐(1→O)(CH2)3S(CH2)6SH, β‐D‐Glcp‐NAc‐(1→3)‐α‐L‐Fucp‐(1→O)(CH2)3S(CH2)6SH, and β‐D‐Glcp3S‐(1→3)‐α‐L‐Fucp‐(1→O)(CH2)3S(CH2)6SH is presented. Such supramolecular structures are excellent probes for studying carbohydrate−carbohydrate interactions by transmission electron microscopy, thereby generating information on the molecular level about the role of different functionalities in the self‐recognition process. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Molecular Fingerprinting of Carbohydrate Structure Phenotypes of Three Porifera Proteoglycan-like Glyconectins

Glyconectins (GNs) represent a new class of proteoglycan-like cell adhesion and recognition molecules found in several Porifera species. Physico-chemical properties of GN carbohydrate moieties, such as size, composition, and resistance to most glycosaminoglycan-degrading enzymes, distinguish them from any other type of known glycoproteins. The molecular mechanism of GN-mediated self/non-self discrimination function is based on highly species-specific and Ca(2+)-dependent GN to GN associations that approach the selectivity of the evolutionarily advanced immunoglobulin superfamily. Carbohydrates of glyconectins 1, 2, and 3 are essential for species-specific auto-aggregation properties in three respective Porifera species. To obtain a structural insight into the molecular mechanisms, we performed carbohydrate structural analyses of glyconectins isolated from the three sponge model systems, Microciona prolifera (GN1), Halichondria panicea (GN2), and Cliona celata (GN3). The glycan content of all three GNs ranged between 40 and 60% of their total mass. Our approach using sequential and selective chemical degradation of GN glycans and subsequent mass spectrometric and NMR analyses revealed that each glyconectin presents novel and highly species-specific carbohydrate sequences. All three GNs include distinct acid-resistant and acid-labile carbohydrate domains, the latter composed of novel repetitive units. We have sequenced four short sulfated and one pyruvilated unit in GN1, eight larger and branched pyruvilated oligosaccharides in GN2, which represent a heterogeneous but related family of structures, and four sulfated units in GN3.

Molecular Recognition between Glyconectins as an Adhesion Self-assembly Pathway to Multicellularity

The appearance of multicellular forms of life has been tightly coupled to the ability of an organism to retain its own anatomical integrity and to distinguish self from non-self. Large glycoconjugates, which make up the outermost cell surface layer of all Metazoans, are the primary candidates for the primordial adhesion and recognition functions in biological self-assembly systems. Atomic force microscopy experiments demonstrated that the binding strength between a single pair of Porifera cell surface glyconectin 1 glycoconjugates from Microciona prolifera can hold the weight of 1600 cells, proving their adhesion functions. Here, measurement of molecular self-recognition of glyconectins (GNs) purified from three Porifera species was used as an experimental model for primordial xenogeneic self/non-self discrimination. Physicochemical and biochemical characterization of the three glyconectins, their glycans, and peptides using gel electrophoresis, ultracentrifugation, NMR, mass spectrometry, glycosaminoglycan-degrading enzyme treatment, amino acid and carbohydrate analyses, and peptide mapping showed that GNs define a new family of proteoglycan-like molecules exhibiting species-specific structures with complex and repetitive acidic carbohydrate motives different from the classical proteoglycans and mucins. In functional self-assembly color-coded bead, cell, and blotting assays, glyconectins displayed species-specific recognition and adhesion. Affinity-purified monospecific polyclonal antibodies prepared against GN1, -2, and -3 glycans selectively inhibited cell adhesion of the respective sponge species. These results together with species-specific coaggregation of GN carbohydrate-coated beads with cells showed that GN glycans are functional in cell recognition and adhesion. The specificity of carbohydrate-mediated homophilic GN interactions in Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily. Xenoselectivity of primordial glyconectin to glyconectin recognition may be a new paradigm in the self-assembly and non-self discrimination pathway of cellular adhesion leading to multicellularity.