Glycobiology Advance Access originally published online on May 4, 2005
Glycobiology 2005 15(9):849-860; doi:10.1093/glycob/cwi072
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Production and characterization of a phage-display recombinant antibody against carrageenans: evidence for the recognition of a secondary structure of carrageenan chains present in red algae tissues
2 URBV, University of Namur, Rue de Bruxelles 61, 5000 Namur, Belgium; and 3 UMR7139 CNRS-Goëmar-UPMC «Végétaux Marins et Biomolécules», Station Biologique de Roscoff, Pl. G.Teissier29, 680 Roscoff, France
1 To whom correspondence should be addressed; e-mail: pierre.vancutsem{at}fundp.ac.be
Received on January 12, 2005; revised on April 6, 2005; accepted on April 30, 2005
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Abstract |
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We report the isolation, for the first time by phage display, of a scFv recombinant antibody called B3 directed against carrageenans, the major sulphated polysaccharides of red seaweeds. Immunoassays were used to characterize the binding of B3 antibodies toward the three main carrageenan forms (
, 
, and 
) differing by their sulfonic ester content and the presence of 3,6-anhydrogalactose. In enzyme-linked immunoadsorbent assay (ELISA), B3 soluble scFv showed a high reactivity towards -carrageenan at any titer but, at high titer only, recognized also the highly sulfated -form. Surface-adsorbed -polymers were only recognized in presence of poly-L-lysine (PLL). The replacement of Na+ ions by K+ in the buffers had no effect on -polymer detection but increased the binding of B3 antibodies toward both - and -carrageenans, whereas addition of Ca2+ decreased sharply the recognition of the -form. In competitive assays, low titer B3 soluble scFv showed a >> selectivity and recognized a mixture of -oligomers with degrees of polymerization between 4 and 18 but not sub-fractions of 4 or 6 residues long. We suggest therefore that the B3 epitope could consist of a helical conformation of carrageenan chains. Immunofluorescence microscopy showed that, amongst other red algae, Chondrus gametophyte (containing -chains) was strongly recognized by B3 scFv whereas sporophytic tissues rich in -carrageenans were not, assessing the preference of this probe for -carrageenans in situ. The high potential of the B3 recombinant probe is discussed. Key words: carrageenan / helical conformation / immunolabeling / phage display / red algae
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Introduction |
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TopAbstractIntroductionResultsDiscussionConclusionMaterials and methodsAcknowledgmentsReferences |
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Carrageenans are a family of sulfated polysaccharides mainly found in red seaweeds (Rhodophycea). They consist of repeating disaccharide units of 3-linked ß-D-galactopyranose (Galp) and 4-linked
-D-galactopyranose. Three main structural groups are defined based on the presence or absence of -D-anhydrogalactose in place of -D-galactopyranose and on the number and position of sulfate groups (Figure 1): - and -carrageenans have in common the repeating sequence [Æ3)-ß-D-Galp-4-sulphate-(1Æ4) 3,6-anhydro--D-Galp (1Æ]n, with the anhydrogalactose residue sulfated on position 2 in . -Carrageenans have no anhydrogalactose residue and are characterized by the repeating unit [Æ3)-ß-D-Galp-2-sulphate-(1Æ4)--D-Galp-2, 6-sulphate (1Æ]n (Tombs and Harding, 1998
). It is worth to note that these structures are idealized views of carrageenans which occur naturally as complex heteropolymers containing other carbohydrate residues and substituents (van de Velde et al., 2002a).
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Because of the 1C4-conformation of their 3,6-anhydrogalactose units, - and -carrageenans are able to undergo coil to helix conformational transition, the subsequent association of helices leading to their gelation. It is now largely accepted that this disorder–order transition corresponds to the formation of double helices occurring, amongst other factors, under suitable ionic conditions. In the presence of monovalent ions such as potassium, -carrageenans form double helices which in turn further aggregate, facilitating the formation of brittle, hard, and strong gels. This helix–helix aggregation process is, however, prevented by iodide ions which stabilize the double helix conformation of -chains (Grasdalen and Smidsröd, 1981). In the case of -carrageenans, the coil-to-helix transition is not cation (monovalent) or anion specific but seems to depend mainly on ionic strength. Compared with -carrageenans, the double helical conformation of -carrageenans is considered to be thermodynamically more stable and less susceptible to aggregation, leading to the formation of soft and weak gels (Austen et al., 1985). However, calcium ions are able to promote aggregation of -carrageenan helices, forming therefore transparent and rigid gels (Yugushi et al., 2003). In contrast, the absence of a 3,6-anhydro bridge in -carrageenans prevents the formation of helical strands and the gelation of these polymers which are always in the random coil conformation (van de Velde and De Ruiter, 2002). These properties are largely exploited in the food industry where carrageenans are extensively used as thickeners, gelling agents, and stabilizers of emulsions. Because of their biocompatibility and noncytotoxicity, carrageenans are also particularly suitable for microencapsulation and drug delivery in the pharmaceutical domain (Tombs and Harding, 1998).
The carrageenan backbone is believed to be synthesized in the Golgi apparatus. - and -carrageenans are synthesized as precursors, respectively called µ and 
, which lack the 3,6-anhydro bridge. The formation of these bridges is catalyzed by the action of sulfohydrolases acting in the cell wall. Mainly found in the cell wall and/or intercellular matrix of red algae, carrageenans are thought to play a role as water reservoir to prevent desiccation and to give the seaweed the flexible structure required to accommodate mechanical stresses encountered in the marine environment. Whereas it is well known that some seaweed genera biosynthesize different carrageenan types in their alternating life stage, the physiological significance of such variations in the composition of the extracellular matrix is still unclear (van de Velde and De Ruiter, 2002).
Carrageenans are bioactive compounds that exhibit a regulatory activity on algal and plant cells: oligomers modulate the virulence of a green algal endophytic pathogen of a red alga (Bouarab et al., 1999), they enhance stress-induced microspore embryogenesis (Lemonnier-Le Penhuizic et al., 2001) and elicit laminarinase activity in Rubus fruticosus cells (Patier et al., 1995). As sulfated polymers, they present a structural homology with glycosaminoglycans and have also many biological properties on animal cells: anticoagulant and antithrombic effects have been reported (Carlucci et al., 1997), as well as antiviral activities, including the inhibition of viruses such as human immunodeficiency virus-1 (Pearce-Pratt and Phillips, 1996; Witvrouw and De Clercq, 1997; Schaeffer and Krylov, 2000; Yamada et al., 2000) which is exploited for the design of new drugs (Vlieghe et al., 2002). These polymers were also shown to interfere with the adhesion (Liu et al., 2000) or the proliferation (Hoffman et al., 1995) of carcinoma cell lines.
Very few specific probes are available to carrageenans. Some antibodies have been obtained and used for localization studies or analysis of food products (Vreeland et al., 1992; Haines and Patel, 1997; Roberts and Quemener, 1999), but none of them were extensively characterized and their application for the detection and identification of carrageenans is still very limited. Because polysaccharides are often poor immunogens, antibodies specific for these polymers are difficult to obtain and the development of high-titre and precisely characterized antibodies to plant polysaccharides is still a priority. To overcome immunization and production problems of monoclonal antibodies, we used the phage display technology to produce probes against carrageenans.
Antibody phage-display libraries consist of genes encoding the antigen binding portions of antibodies (V-genes) fused in frame with a filamentous phage coat protein gene (M13 protein gene III). Each resulting antibody fragment (e.g., scFv) is displayed as a fusion product on the surface of one phage particle that carries the gene encoding that antibody fragment, allowing therefore further manipulations to improve antibody specificity and/or affinity (Nissim et al., 1994).
Phage antibodies directed against carbohydrate moieties such as rhamnogalacturonan II and Lewisx antigens have been successfully isolated from immune libraries, that is, libraries constructed for each chosen antigen by cloning the antibody genes from immunized donors (Williams et al., 1996; Mao et al., 1999). Recombinant antibodies were also obtained against heparan sulfates (van Kuppevelt et al., 1998; Dennissen et al., 2002) and deesterified homogalacturonans (Willats et al., 1999) from synthetic libraries built by cloning artificial antibody variable domain genes resulting from the in vitro assembly of antibody variable gene segments [often modified in their complementary-determining regions (CDRs)]. Such libraries, available commercially or through academic laboratories, are often large (up to 1010 binders) and can be used for the selection of antibodies against almost any given antigen in a few weeks. In this work, we have exploited the so-called Griffin-1 library (H. Griffin, MRC, Cambridge, UK), to select antibodies specific for -carrageenans, polymers against which several attempts to generate antibodies were unsuccessful in the past (Dininno and McCandless, 1978). One of these antibodies, called B3, was extensively characterized and used for the in situ localization of carrageenan epitopes within seaweed tissues.
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Results |
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Affinity selection of anticarrageenan recombinant antibodies
Phage antibodies specific for carrageenans were isolated from the Griffin-1 library containing human scFv fragments that result from the recloning into the phagemid vector pHEN2 of variable antibody region (VH and VL) genes with synthetic random CDR3 segments, originally contained in lox library vectors (Griffiths et al., 1994
). Five rounds of panning were performed against -carrageenans and casein hydrolysate (control). A 640-fold enrichment in the titer of -carrageenan-eluted phages was observed from the second to the fifth round whereas the amount of phages eluted from the control antigen decreased continuously until no phage still bound to casein hydrolysate at the fourth and fifth rounds. A polyclonal phage enzyme-linked immunoadsorbent assay (ELISA) with the original and enriched phage libraries showed a strong answer against -carrageenan from phage preparations of the fourth and fifth rounds of panning (Figure 2), without any significant detection of the control antigen.
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Monoclonal phage antibody preparations derived from 120 colonies randomly picked from the fourth and fifth enriched phagemid libraries were screened against -carrageenans and casein hydrolysate as control antigen. After the fourth round of selection 25 phage clones (42%) showed absorbance values greater than 1.5, and values greater than 2 were observed for 12 clones (20%). The proportion of such middle- and high-response clones increased respectively to 75% (45 clones) and 40% (24 clones) after the fifth round of panning. None of these clones showed any significant reactivity to casein hydrolysate. Fingerprinting analysis of 30 high-response clones revealed the existence of at least five different phage antibodies (data not shown). These phages were used to infect Escherichia coli strain HB2151 from which crude periplasmic extracts were prepared and analyzed for the presence of recombinant antibodies by western blot labeling using antibodies directed against the C-terminal c-myc tag of the fusion protein. Only one of these extracts (clone B3) showed a very strong reactivity toward -carrageenans in ELISA and was therefore kept for further work. As shown in Figure 3, a single band with a molecular weight (MW) slightly below 35 kDa was detected in periplasmic fractions obtained from B3 cultures, which is in agreement with the theoretical MW of 30 kDa calculated for the B3 scFv. In contrast, no scFv was detected in culture supernatants. B3 scFv purified by Ni2+ –iminodiacetic acid (IDA) chromatography were also prepared and analyzed by western blot (Figure 3): in addition to the major band at 30 kDa, the anti-c-myc antibody labeled minor proteins of higher MW, corresponding probably to scFv aggregates, such as dimers, known to occur in such concentrated antibody preparations (MacKenzie and To, 1998). Used as either purified or crude periplasmic preparations, B3 scFv gave similar anticarrageenan answers (not shown). Most of our assays were therefore performed with easily obtained and rapidly prepared crude periplasmic extracts.
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Characterization of the B3 anticarrageenan antibodies
B3 soluble scFv are specific for carrageenans
In ELISA tests performed with low polymer concentration (25 µg/mL) (Figure 4A), the B3 scFv bound only -carrageenan and neither nor . No significant cross-reactivity was observed with agarose, a closely related molecule characterized by the repeating disaccharide sequence ((3)-ß-D-Galp-(1Æ4) 3,6 anhydro--L-Galp (1Æ)n], nor with moderately sulfated polymers such as dextran sulfate, chondroitin 4-sulfate, keratan sulfate, and heparan sulfate (not shown) or highly sulfated polysaccharides such as pentosan polysulfate, heparin, and fucoidan which present three to four sulfates per disaccharide repeating unit. Other negatively charged polysaccharides such as polygalacturonic and polyguluronic acids were not recognized by the antibodies, neither was polymannuronic acid (not shown). Amongst all these polymers, only the highly sulfated fucoidan, polysaccharide made of 3- and 4-linked -L-fucopyranose 2-sulfate residues with additional sulfate groups on some 3-linked fucopyranoses (Bilan et al., 2002), was found to be significantly recognized by the antibodies under conditions of both high polysaccharide coating concentration and high antibody titer (Figure 4B). This nonspecific binding decreased upon polymer and/or antibody dilutions whereas, at the same dilutions, detection of -carrageenans remained particularly high.
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B3 soluble scFv show a different reactivity towards carrageenans
At high concentration of periplasmic extracts, B3 soluble scFv showed a similar binding to - and -carrageenans although, at lower titers (40x dilution), the recognition of the -form dropped sharply as compared to (Figure 5). In all cases, the samples were the less recognized. To rule out any effect of microplate coating efficiency by the three carrageenans, competitive assays were performed with -carrageenans as standard solid-phase antigens (Figure 6). Whereas about 50 µg/mL of -carrageenans were needed for 50% inhibition of B3 scFv binding to immobilized -carrageenans, -, and -carrageenans had 50% inhibitory concentrations (IC50) of 
9 and 0.7 µg/mL. The effective binding of B3 antibodies to -carrageenans in solution contrasted with the poor recognition of the same -polymers when adsorbed onto Maxisorp plates.
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Surface adsorbed -carrageenans are only recognized in presence of poly-L-lysine
Precoating of the microwells with positively charged poly-L-lysine (PLL) strongly increased the recognition of - and especially -carrageenan by the soluble B3 scFv (Figure 7). In presence of PLL, a maximal detection of -carrageenan by the scFv occurred for concentrations at least 10 times lower than in absence of PLL. -carrageenan could virtually not be detected by B3 scFv without precoating of the microwells by PLL. The presence of PLL had very little effect on the recognition of -carrageenan by the soluble scFv. Amongst other sulfated polymers, only fucoidan and pentosan polysulfate were slightly detected by B3 antibodies when adsorbed on PLL-coated microwells (data not shown).
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Ionic conditions modify the binding of recombinant antibodies to carrageenans
Whereas B3 soluble antibodies show a similar detection of carrageenans in Tris- and phosphate-based buffers containing NaCl (Figure 8), the replacement of Na+ by K+ ions promoted the binding of B3 scFv to both - and -carrageenans, but had no incidence on -polymer recognition. A sharp decrease of -carrageenans recognition resulted from the addition of calcium ions to the Tris–NaCl buffer (Figure 8).
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Determination of the B3 epitope structure
The >> binding preference of monomeric B3 scFv in competition assays rises questions about the length and nature of the epitope recognized. The B3 soluble scFv binding to immobilized -carrageenans was prevented by competing hydrolysates of -carrageenan and to a lesser extent -carrageenan oligomer mixtures with lengths ranging from 4 to 18 residues (Figure 9). Hydrolysis products of -carrageenan with a similar range of length, as well as purified - and -oligomers of 4 or 6 residues long were unable to prevent binding of the B3 scFv to the -carrageenans.
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Analysis of B3 sequence
Comparison of B3 sequence (Figure 10) with the V BASE germline directory revealed that the VH chain belonged to VH1 family (segment DP-25) whereas the VL domain was derived from V1 (segment DPK-1). VH CDR3 contained only four amino acids among which one positively charged (R). Respectively one and two positive residues were also found in VH CDR1 and CDR2 but none in the CDRs of VL. Negative amino acids were only present in the CDRs of VL. Analysis of the B3 sequence against a ‘Conserved domain database’ (http://www.ncbi.nih.gov/StructureG) indicated a high homology of the B3 VL, and especially of its CDRs, with a portion of the light chain of a neutralizing human antibody binding to a HIV gp120 epitope (Kwong et al., 1998). However, the gp120 epitope recognized by this antibody is different from the V3 loop of gp120 known to bind carrageenans (Witvrouw and De Clercq, 1997).
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-Carrageenan labeling of red algae cross-sections
-Carrageenans were located within the thallus tissues of carrageenophytes by a three-step immunofluorescence procedure using B3 soluble scFv (primary) antibodies, mouse anti-c-myc (secondary) antibodies, and Alexa 488 labeled goat anti-mouse (tertiary) antibodies (Figure 11). The Chondrus crispus cell wall of the gametophyte well known to contain -carrageenan was used as positive control. It was heavily labeled on both cell walls and intercellular matrix (Figure 11, A2–A4). The -carrageenan deficient cell wall of the C. crispus sporophyte was weakly labeled and one could only observe fluorescence on the internal part of the thallus (Figure 11, B2–B4).
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The B3 epitope was abundantly detected in the cell walls of other red algae belonging to the following species: Mastocarpus stellatus (Figure 11, C2–C4), Chondracantus acicularis (Figure 11, D2–D4), Catalena opuntia (Figure 11, E2–E3), Plocamium cartilagineum (Figure 11, F2–F3), and Furcellaria fastigiata (Figure 11, G2–G4). If present, the intercellular matrix was also strongly labeled by the antibodies, as observed on the central region of Furcellaria thallus, around closely packed round-shaped cells (Figure11, G2–G3) and throughout the Mastocarpus thallus, surrounding elongated and rounded cells (Figure 11, C2–C4). The cuticle, in contact with the external medium, was also strongly recognized (Figure 11, C3, D3, E3, and F2), as well as sorts of buddings occurring on Catalena surface. The specificity of the labeling on algal tissues was assayed using B3 scFv preincubated with -carrageenans (example illustrated in Figure 11, F4). Similar observations were achieved with the pretreated -carrageenase cross-sections performed on all the algal specimens (results not shown). No labeling could be observed when experiments were conducted on untreated cross-sections using the secondary and tertiary antibodies (Figure 11, E4).
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Discussion |
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TopAbstractIntroductionResultsDiscussionConclusionMaterials and methodsAcknowledgmentsReferences |
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We have generated phage display antibodies that specifically bind to carrageenans. To our knowledge, this is the first time recombinant antibodies directed against polysaccharides from algal origin were isolated by a phage display approach, in this case using the Griffin-1 library, also known as ‘Human synthetic VH + VL scFv library’. Other recombinant antibodies directed against polysaccharidic antigens (van Kuppevelt et al., 1998
; Willats et al., 1999) were isolated from another ‘single pot’ library, the ‘synthetic scFv Library (#1)’ (Nissim et al., 1994). In the context of the production of specific probes against plant or algal polysaccharides, which are often poor immunogens, the phage display approach using large naïve libraries presents the major advantage of allowing the isolation of antibodies in a few weeks, bypassing limitations because of immunization or library construction steps (Willats, 2002).
Characterization of the B3 recombinant antibody
B3 soluble scFv showed a strong preference for immobilized -carrageenans but also strongly recognized the -form at high titers (i.e., periplasmic extracts above 100 µg of proteins/ mL or Ni2+–IDA purified scFv). However, the binding to these highly sulfated -polymers disappeared at lower antibody titers, although their strong preference for -carrageenans was still maintained. Similarly, but to a much lower extent (Figure 4B), high titer B3 antibodies were also found to bind to fucoidan, another highly sulfated polysaccharide. As for the -form recognition, this cross-reactivity decreased upon antibody and/or polymer dilution. Accordingly, the binding of B3 scFv on the -polymer could result from its high degree of sulfation and possibly reflect the initial stage of antibody-epitope recognition, driven by electrostatic interactions between sulfate groups and the positively charged amino acids found in the CDRs of the B3 antibody VH. The importance of charge interactions for B3 antibody–antigen binding is further highlighted by the observation that an increase of the pH in the ELISA working buffer (from 7.2 to 8.2) resulted in a sharp decrease of -carrageenan recognition (results not shown).
In consequence, this -polymer recognition led us to determine, for each batch of antibody preparation, the scFv dilution to be used to optimize the discrimination between the - and the -forms.
Competition ELISA assays confirmed the preference of B3 soluble scFv for -carrageenan: about 0.7 µg/mL of this polymer was required for 50% inhibition of antibody binding. IC50 values obtained with soluble scFv indicate that competition was about 10 and 70 times lower with - and -polymers (Figure 6). This selectivity preference of B3 for - over - and -carrageenans is largely higher than what was mentioned for the only other anti--probe described, the -related 4D12 monoclonal antibody, which exhibited a 2.9 and a 1.9 preference for the -form over the - and -forms, respectively (Vreeland et al., 1992). The heterogeneity of carrageenan molecular weight did not allow us to determine antibody affinity.
To further elucidate the nature of the B3 epitope, the reactivity of the recombinant antibodies with the three carrageenan forms was evaluated under the highlight of the conformational changes undergone by carrageenans in solution. In comparison with the huge literature available upon the rheological behavior of carrageenans under gelling conditions, that is, polymer concentrations around 5 g/L (Tombs and Harding, 1998), there is little information regarding their conformational changes occurring under nongelling conditions such as those used in our assays (i.e., polymer concentration lower than 500 µg/mL and 100 mM NaCl). Interestingly, light scattering studies performed in similar conditions have shown that -carrageenans undergo conformational transition from coil to helices (Vanneste et al., 1994), whereas -carrageenans were found to stay mostly in a coil-like conformation (Slootmaekers et al., 1991). We therefore suggest that our B3 antibody would preferentially recognize a helical conformation of carrageenans, mostly adopted by the -form under our working conditions. Accordingly, the lower recognition of the -carrageenans by B3 antibodies in competitive ELISA could simply be due to the lower ability of these polymers to form helices in the Na-based phosphate-buffered saline (PBS) buffer, their adsorption onto the microwells preventing them from any conformational change and, in consequence, from any recognition by B3 antibodies. The positive effect of PLL on the recognition of immobilized -carrageenans by B3 antibodies is probably not caused by an increased -polymer adsorption in the microwells, because these carrageenans were efficiently recognized by other phage antibodies in absence of PLL precoating (data not shown). As demonstrated for -carrageenans (Girod et al., 2004), -carrageenans were also shown to form polyelectrolyte complexes with PLL (Domard and Rinaudo, 1981). We can therefore reasonably assume that PLL molecules coated in the microwells could drive conformational changes of -chains, allowing them to adopt helical conformation and, in consequence, to be recognized by B3 antibodies as well as -ones. However, PLL is apparently not able to force -carrageenans to form the helices recognized by the B3 soluble scFv. The slight binding of B3 scFv to the -form in solution would not be associated to any conformational change of this polymer, considered to exist under a stiff coil conformation (Tombs and Harding, 1998), but would instead result from its high degree of sulfation, as above mentioned. Our observation of a similar slight recognition of largely sulfated fucoidan and pentosan polysulfate in PLL precoated wells seems in agreement with such an assumption.
Our hypothesis that B3 scFv epitope could correspond to -carrageenan helices raises questions on the nature of this helical conformation potentially recognized by the antibodies. Even if under gelling conditions, X-ray diffraction studies (helical structures available at PDB—http://www.rcsb.org/pdb) have led to a widely accepted model in which - and -carrageenan helices are formed by two chains wrapping around one another (double-helix) (Anderson et al., 1969), this is disputed by some authors who rather favor a model in which an helix is formed from one strand wrapping upon itself (single-helix) (Smidsrød et al., 1980). There seem to be strong evidence that, in nongelling conditions, the occurrence of the ordered helical state was not accompanied by intermolecular chain dimerization (Vanneste et al., 1994; Viebke et al., 1995; Reynaers, 2003), ruling out the possibility of the existence of intermolecular double-stranded helices in that case. However, the exact nature of this monomolecular ordered helical conformation is still unclear: conflicting evidence support either a local double-helix generated intramolecularly in an antiparallel (hairpin-like) or a parallel (cyclic-like) fashion (Stokke et al., 1993; Viebke et al., 1995) or a single-stranded helix (Reynaers, 2003). The B3 conformational epitope could thus correspond to such helical forms of -carrageenans, but our data do not allow us to favor one model upon another.
It is interesting to note that the helical epitope assumption is compatible with our findings that calcium ions, known for their ability to induce aggregation of -carrageenan helices, decreased sharply the recognition of -carrageenans by B3 antibodies. Similarly, K+ ions, known to aggregate preformed -carrageenan helices, were unable to promote the recognition of the -form (also observed in PBS-based buffers, not shown), which excludes the possibility of any binding of the B3 antibodies to aggregated -carrageenan helices. Because of the denaturation of the NaI solubilized ELISA reagents, we were not able to check the recognition by the antibodies of NaI-stabilized -carrageenan helices.
Finally, competition assays performed with carrageenan hydrolysis products and purified oligomers give further arguments in favor of the recognition of a conformational epitope by B3 antibodies. Evidence of a size-dependent conformational transition from coil to helix is provided for -oligomers of degree of polymerization on monomer basis (DP) 
8 (i.e., at least four dimer units) in presence of potassium but not sodium (Rochas et al., 1983). The 100 mM NaCl concentration used in our experiments must have prevented helix formation by the -oligomers with lengths up to 18 residues, which is confirmed by their absence of recognition by the B3 antibodies. The - and the -forms have a very similar chemical structure, the anhydro bridges conferring them flexibility, but the -oligomers undergo a size-dependent coil–helix transition even in presence of sodium salts, as shown previously for the polymer chain. The critical length of -sequence allowing a coil to double helix transition includes about eight repeating units of -carrageenans (i.e. 16 sugar monomers) (van de Velde et al., 2002b). This is consistent with the lack of competition we observed for isolated -oligomers of DP 4 and 6 and the effective competition obtained with -oligomer mixtures containing fragments with lengths up to 18 residues. DP 16 is much larger than the DP of 4–6 sugar residues of a typical sequential epitope, which would confirm the conformational nature of the B3 epitope. As it is known from the literature that -polymers never adopt helical conformation, the unexpected strong competitive effect of the enzymatically-produced mixture of -oligomers is more difficult to interpret. In our opinion, it is difficult to consider that such a strong binding of the antibodies to these oligomers could simply result from their higher degree of sulfation. Because the oligomers are certainly much shorter than the 80 nm persistence length estimated for a typical -carrageenan (Reynaers, 2003; to our knowledge, no -value is available), we suggest that the B3 scFv could be able to force these -oligomers to adopt a conformation close to the one spontaneously adopted by -oligomers. This concept of stabilization by an antibody of a molecule under a higher energy conformation is at the basis of the development of catalytic antibodies. The limited availability of pure and characterized - and -oligomers in sufficient amounts to perform ELISA assays did not allow a further characterization of the process.
Localization of -carrageenans in algae
Immunofluorescence localization of the B3 epitope was performed on several red algae species growing on French coasts, Eucheumoids algae being hardly available. A heavy labeling was observed on cell walls of all tested algae samples and, in some cases, also on the matrix found between cells of some species such as Furcellaria and Mastocarpus, which is in agreement with the general distribution pattern of carrageenans in red algal tissues (Gretz et al., 1997). This large distribution of B3 epitope in algal tissues indicates that local intrachain -double helices and, most probably, interchain –double helix associations occur in muro. The dense labeling obtained with B3 antibodies on gametophytic plants of C. crispus is consistent with the fact that these tissues are known to contain /-hybrid carrageenans made of a mixed chain of both - and -units (van de Velde et al., 2001). On the other side, C. crispus sporophytic plants characterized by their production of -carrageenans (van de Velde and De Ruiter, 2002) are only weakly recognized by B3 antibodies. Such a slight binding has been previously found by Vreeland et al. (1992) using their anti- 4D12 monoclonal antibody to label tissues of the -carragenophyte Kappaphycus alvarezii.
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Conclusion |
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TopAbstractIntroductionResultsDiscussionConclusionMaterials and methodsAcknowledgmentsReferences |
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Although phage display is a powerful technique to obtain antibodies against polysaccharides such as carrageenans, the epitope mapping of such probes is still a challenge. Our results suggest that the B3 epitope could consist of a helical conformation characteristic of
-carrageenan chains: the 1C4 conformation of the ß-(1Æ4)-linked galactose unit of -carrageenans is locked by the 3,6-anhydro bridge allowing these polymers to adopt a helical secondary structure which is essential for gel formation by interpenetration of preformed helices belonging to neighboring chains (van de Velde et al., 2002b). The B3 antibodies allowed the detection of the helical conformation of -carrageenan chains in situ, in the cell wall and intercellular matrix of algal tissues.
This probe can be used to characterize carrageenan polysaccharides present in (or extracted from) red algal tissues, as a fast and powerful tool complementary to chemical and physical approaches largely used to analyze carrageenan molecules. It gives the possibility to address, at the ultrastructural level, questions regarding the biosynthesis of carrageenans and the biological importance of the helical conformation of carrageenan chains in situ. Indeed, the process of carrageenan biosynthesis is far from being understood: if there is a general agreement that -carrageenans are synthesized as µ precursors, some authors rather suggest that the biosynthesis of -carrageenan is completed intracellularly (Gretz et al., 1997). It is still not known whether such newly synthesized -carrageenans are able to form helices in the Golgi cisternae or if their ability for conformational changes appears only later, when they are deposited in the cell wall and/or the intercellular matrix. Similarly, the subcellular localization of some structural modifications of -carrageenans interfering directly with their gelation properties, such as the addition of methyl ether groups, has still to be demonstrated.
The B3 probe will help to investigate further the spatial and temporal variations of -carrageenan during development of algal tissues (Vreeland et al., 1992), as well as to monitor the effects of environmental factors on -carrageenan structure and function. The recombinant antibody technology also opens a new and original way to study structure–activity relationships of carbohydrate molecules. As it has been demonstrated recently that algae can be engineered to produce fully active antibodies (Mayfield et al., 2003), one could express the B3 antibody in a red seaweed to interfere with -carrageenan biosynthesis and/or deposition. Largely used in higher plants to investigate the role of specific antigens (De Jaeger et al., 2000), this approach could help generating kinds of wall mutants in the algal field, precious tools to go deeper in the investigation of carrageenan function.
Along with the 2F4 antipolygalacturonic acid monoclonal antibody (Liners et al., 1992), the B3 recombinant scFv is another example of a probe directed against a supramolecular conformation of an anionic polysaccharide. Pectin as well as carrageenans are two acidic plant polysaccharides with biomodulating activities both in the plant and biomedical fields. The 2F4 monoclonal antibody was instrumental in the elucidation of the size dependency of the elicitating activity of pectic oligomers. The B3 recombinant antibody could also be helpful for understanding the biological activity of carrageenan oligomers.
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Polysaccharides
-, -, and -Carrageenans (C 1263, C 4014, and C 3889), neocarratetraose 41, 43-disulfate (N 8136), neocarrahexaose 41, 43, 45-trisulfate (N 8268), dextran sulfate (D4911), chondroitin 4-sulfate (C8529), keratan sulfate (K 3001), heparan sulfate (H4777), heparin (H4784), and fucoidan (F5631) were obtained from Sigma (St. Louis, MO). Unfractionated preparations of -, -, and -oligomers (4–18 residues length), purified fractions of -oligomers of DP 4 and 6, were prepared according to Lemonnier-Le Penhuizic et al. (2001) and checked by NMR.
Selection of anticarrageenan phage antibodies by library panning
Phage display-derived antibodies were selected from the Griffin-1 library kindly provided by the MRC centre (Cambridge, UK) using five rounds of panning against -carrageenan. Thus, three wells of Maxisorp ELISA plates (Nunc, Denmark) were coated overnight at 4°C with 100 µL of a 100 µg/mL -carrageenan solution in PBS (5.84 g NaCl, 4.72 g Na2HPO4, and 2.64 g NaH2PO4.2H2O per liter, pH 7.2). In parallel, three other wells were coated with 100 µL of 1% (w/v) casein (Hammerstein grade, MP Biomedicals, Irvine, CA) in phosphate-buffered saline containing 1% casein hydrolysate (C-PBS) for control panning experiments. Wells were rinsed three times with PBS, blocked by incubation with 250 µL of C-PBS for 2 h at room temperature and rinsed again three times with PBS. Phage suspensions diluted in C-PBS and left for 30 min under gentle shaking at room temperature were added to the wells (4 x 1011 phages per well in a volume of 100 µL) and allowed to stand for 2 h at room temperature. Wells were washed 10 times with PBS containing 0.1% (w/v) Tween 20 (PBS-T) and 10 times with PBS for the first round of selection. For the second and subsequent rounds of selection, the wells were washed 20 times with PBS-T and 20 times with PBS. Bound phages were eluted by a 10 min incubation with 100 µL per well of 100 mM triethylamine and immediately neutralized with 300 µL of 1 M Tris–HCl (pH 7.4). Eluted phages were used to reinfect a fresh culture of TG1 bacteria which were plated on Trypton Yeast Extract medium (15 g bacto-agar, 8 g NaCl, 10 g tryptone, and 5 g yeast extract per liter) containing 2% (w/v) glucose and 100 µg/mL ampicillin and grown overnight at 30°C, allowing the determination of the titers of the -carrageenan-bound («positive») phages and the casein-bound («control») phages, respectively. Colonies of transfected TG1 bacteria from the carrageenan-derived library were scraped from the plates and used to inoculate 2 xTY medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl per liter) containing 2% (w/v) glucose and 100 µg/mL ampicillin (2 xTY-gluc.-amp.). They were infected with M13K07 helper phage (GE Healthcare, Fairfield, CT) and grown overnight at 30°C in a 2 xTY medium containing 100 µg/mL ampicillin and 50 µg/mL kanamycin to produce phage particles which were rescued by polyethylene glycol/NaCl precipitation and used for further rounds of selection.
Monitoring of enriched phage libraries by phage ELISA
Maxisorp ELISA plates were coated with -carrageenan or C-PBS (control wells) and blocked with C-PBS as described above. After three washes with PBS, phage suspensions used for each round of panning (see previous section) were diluted in C-PBS and left for 30 min under gentle shaking at room temperature before being added to the wells (4 x 1011 phages per well in a volume of 100 µL) and allowed to stand for 2 h at room temperature. Unbound phage antibodies were removed by three washes with PBS-T followed by three washes of PBS. Bound phages were detected using 100 µL per well of a horseradish peroxidase-conjugated anti-M13 antibody (GE Healthcare) diluted 1/5000 in C-PBS and incubated for 1 h at room temperature. After washing as described above, the peroxidase activity was detected using 100 µL/well of K-blue substrate® (Neogen Corp., Lansing, MI). The reaction was stopped after 20 min in the dark with 100 µL 1M HCl per well and absorbances were measured at 450 nm.
Screening of monoclonal phage antibodies
Bacteria picked from 120 colonies derived from the last two rounds of selection were individually grown in 100 µL of 2 xTY-gluc.-amp. medium (see second section) in 96-well V-bottom plates (Corning, Corning, NY) overnight at 30°C. Inoculums (20 µL) of this plate were transferred to a second plate containing 200 µL of a M13K07 phage suspension (0.5 109 pfu/mL of 2 xTY-gluc.-amp.) and grown at 37°C for 2 h. After 10 min centrifugation, the supernatants were removed, the pellets were resuspended in 200 µL 2 xTY medium containing 100 µg/mL ampicillin and 50 µg/mL kanamycin and grown overnight at 30°C. Phage supernatants recovered after centrifugation were diluted in C-PBS and left for 30 min under gentle shaking at room temperature before being analyzed by phage-ELISA as previously described.
Analysis of phage-antibody clones
ELISA-positive phages were checked for the presence of full-length inserts by polymerase chain reaction (PCR) on bacteria transferred in a PCR mix containing 100 pmol of the primers LMB3 (5'-CAGGAAACAGCTATGAC-3') and fd-SEQ1 (5'-GAATTTTCTGTATGAGG- 3') by using the following program: 30x (1 min at 94°C, 1 min at 56°C, and 2 min at 72°C) followed by 10 min at 72°C. Fingerprinting was performed by the digestion of the PCR products with MvaI (Roche, Basel, Switzerland) and analysis of the restriction profiles by agarose gel electrophoresis. Clones were sequenced on an automated DNA sequencer (Applied Biosystem, Foster City, CA) by the dideoxy method (Sanger et al., 1977) using plasmid DNA prepared according to standard procedures. V segments of B3 antibodies were identified according to the V BASE germline directory (http://www.mrc-cpe.cam.ac.uk). The nucleotide sequence of the B3 clone can be found in GenBankTM. (accession number AY496269
[GenBank]
).
Expression and purification of scFv fragments and western blot analysis
ELISA positive phages were used to infect E. coli HB2151 and periplasmic extracts of recombinant bacteria were prepared according to the recombinant phage antibody system protocol (RPAS, GE Healthcare). The scFv present in these extracts were purified on a Ni2+–IDA column according to the manufacturer instructions (Novagen, Darmstadt, Germany). The protein content of the crude extracts and the purified fractions was measured with BioRad’s protein assay reagent using bovine serum albumin as standard. Culture supernatants, periplasmic extracts, and purified scFv were analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blotting using Trans-blot Semi-dry transfer system (Bio-Rad) at 15 V for 30 min. The PVDF membrane was blocked for 1 h at room temperature with 5% low-fat dried milk in Tris Buffered Saline (TBST; Tris–HCl 20 mM, NaCl 137 mM, pH 7.6 containing 0.1% Tween 20) and, after 3 washes with TBST, was incubated overnight at 4°C with anti-c-myc tag antibodies (Invitrogen, Carlsbad, CA) diluted 1/1000 in TBST. After 3 rinses with TBST, the membrane was incubated for 1 h at room temperature with alkaline phosphatase-conjugated anti-mouse antibodies, (Sigma, St. Louis, MO) diluted 1/15000 in TBST. The blot was washed 3 times in TBST and developed using 5-bromo-4-chloro-3-indolyl phosphate–p-nitroblue tetrazolium chloride (BCIP–NBT) (Roche).
Evaluation of the binding specificity of B3 scFv by ELISA
Reactivity of B3 scFv toward different polymers was evaluated by scFv ELISA, modified from the phage ELISA as follows: crude periplasmic extracts containing B3 scFv were diluted in C-PBS and left for 30 min under gentle shaking at room temperature before being added to polymer coated wells (unless otherwise stated, wells were coated with 100 µL of a 100 µg/mL solution of each polymer). Bound scFv were detected using a horseradish peroxidase-conjugated anti-c-myc antibody (Invitrogen) diluted 1/1000 in C-PBS. Differences in plate binding properties between carrageenan types were evaluated by addition of a precoating step of 1 h at room temperature with 100 µL per well of PLL (P 1399, Sigma) (0.05 mg/mL in H2O) and 3 washes with PBS before the application of carrageenans. The effect of salts on carrageenan recognition by the antibodies was assessed by scFv ELISA, using the following buffers at each step of the assay: Tris–NaCl/Tris–KCl (20 mM Tris–HCl, pH 7.2 containing 100 mM NaCl or 100 mM KCl); Tris–NaCl–CaCl2 (20 mM Tris–HCl, pH 7.2 containing 100 mM NaCl and 5 mM CaCl2); and control buffer PBS-Na (33 mM Na2HPO4, 17 mM NaH2PO4. 2H2O, pH 7.2, 100 mM NaCl). Competition assays based on scFv ELISA were performed by incubation of the periplasmic extracts containing soluble B3 scFv with different concentrations of carrageenans or oligo-carrageenans in C-PBS during 30 min under gentle shaking at room temperature, followed by transfer to plates (100 µL/well) coated with -carrageenans used as reference antigens. The other steps of the assays were carried on as described above. Positive controls, with no oligo- or polymers as competitors, were included in the assays. The amount of each competitor needed to reduce the antibody binding by 50% (IC50) was calculated by comparison with the positive control reaction.
Tissue preparation and immunolabeling procedure
Samples of C. crispus (gametophyte and sporophyte), M. stellatus, C. acicularis, C. opuntia, P. cartilagineum, and F. fastigiata were collected on French coasts and thallus pieces were processed for microscopy essentially as described by Vreeland et al. (1992): after a fixation step of 1 h at 4°C with 2% formaldehyde and 2% glutaraldehyde in filter sterilized sea water, the thallus pieces were fixed in a renewed solution for an additional 12 h at 4°C. They were rinsed twice with sea water at 4°C for 20 min each rinse and were dehydrated at room temperature (1 h each step) through 20, 30, and 40% ethanol in sea water and 50, 60, 70, and 80% ethanol in deionized water. After a night in 90% ethanol and 3 h in 100% ethanol, they were infiltrated by placing them successively in 2:1, 1:1, and 1:2 (v : v) ratio of absolute ethanol: LR White resin (London Resin Co Ltd, Theale, UK), under rotation at room temperature (1–4 h each step). They were transferred in fresh resin and left overnight at 4°C under rotation. The LR White solution was changed and the polymerization was performed for 6 h at 60°C. Transverse semi-thin sections (1–2 µm) were cut with a pyramitome 11800 (LKB), collected onto microscope slides on a drop of water, and heat-fixed to the slides overnight at 40°C. Representative sections were stained with 0.2% (w/v) toluidine blue in water for 30 s and rinsed with water.
Thallus sections were immunolabeled as follows: after a 1 h blocking step at room temperature with C-PBS, the sections were incubated overnight at 4°C with crude periplasmic extracts containing B3 scFv (17.5 µg of proteins/ml), diluted up to 20 times in C-PBS. B3 antibody binding was detected by incubation with a mouse anti-c-myc tag antibody (Invitrogen) followed by an Alexa 488-labeled goat anti-mouse IgG (Molecular Probes, Carlsbad, CA). Both antibodies were used at a 100-fold dilution in C-PBS and incubated for 1 h at room temperature. Following each incubation with antibody, the sections were washed with PBS-T (3 x 5 min) and PBS (3 x 5 min). Sections were finally mounted with Mowiol and examined on a Zeiss microscope equipped with epifluorescence. Micrographs were taken on 400ASA color film (Kodak). The labeling specificity was assessed by (a) omission of the recombinant antibody and (b) preincubation of the recombinant antibody with the -carrageenan form (100 µg/mL, 1 h at room temperature) prior to immunolabeling.
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We are grateful to the MRC (Cambridge, United Kingdom) for providing the phage display library and to Mrs. C. Devignon (Unité Interfacultaire de Microscopie Electronique, University of Namur, Belgium) for her precious assistance in light microscopy. We also thank Drs. O. Duterme (FUSAGx, Gembloux, Belgium), G. De Jaeger (VIB, Gent, Belgium), and X. DeBolle (University of Namur, Belgium) for useful advice in phage display and Prof. M. Rusnati (University of Brescia, Italy) for the kind gift of pentosan polysulfate. We gratefully acknowledge the support of the Région Wallonne (DGTRE), Belgium, to this work.
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Abbreviations |
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CDRs, complementary-determining regions; C-PBS, phosphate-buffered saline containing 1% casein hydrolysate; DP, degree of polymerization on monomer basis; Galp, 3-linked ß-D-galactopyranose; IC50, 50% inhibitory concentrations; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PLL, poly-L-lysine.
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