Thermodynamic studies on the interaction of water-soluble porphyrins with the glucose/mannose-specific lectin from garden pea (Pisum sativum)

Due to the application of porphyrins as photosensitizers in photodynamic therapy to treat cancer, and the ability of some lectins to preferentially recognize tumor cells, studies on the interaction of porphyrins with lectins are of considerable interest. Here we report thermodynamic studies on the interaction of several free-base and metallo-porphyrins with pea (Pisum sativum) lectin (PSL). Association constants (Ka) were obtained by absorption titrations by monitoring changes in the Soret band of the porphyrins and the Ka values obtained for various porphyrins at different temperatures are in the range of 1.0 x 10(4) to 8.0 x 10(4) M(-1). Both cationic and anionic porphyrins were found to bind to PSL with comparable affinity. Presence of 0.1 M methyl-alpha-D-mannopyranoside--a carbohydrate ligand that is specifically recognised by PSL--did not affect the binding significantly, suggesting that porphyrin and sugar bind at different sites on the lectin. From the temperature dependence of the Ka values, the thermodynamic parameters, change in enthalpy and change in entropy associated with the binding process were estimated. These values were found to be in the range: delthaH degree = -95.4 to -33.9 kJ x mol(-1) and deltaS degree = -237.2 to -32.2 J x mol(-1) x K(-1), indicating that porphyrin binding to pea lectin is driven largely by enthalpic forces with the entropic contribution being negative. Enthalpy-entropy compensation was observed in the interaction of different porphyrins to PSL, with the exception of meso-tetra-(4-sulfonatophenyl)porphyrinato zinc(II), emphasizing the role of water structure in the overall binding process. Circular dichroism and differential scanning calorimetric studies indicate that while porphyrin binding does not induce significant changes in the lectin structure and thermal stability, carbohydrate binding induces moderate changes in the tertiary structure of the protein and also increases its thermal unfolding temperature and the enthalpy of the unfolding transition.     

Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin.

The interaction of several metallo-porphyrins with the galactose-specific lectin from Trichosanthes cucumeirna (TCSL) has been investigated. Difference absorption spectroscopy revealed that significant changes occur in the Soret band region of the porphyrins upon binding to TCSL and these changes have been monitored to obtain association constants (Ka) and stoichiometry of binding (n). The dimeric lectin binds two porphyrin molecules and the presence of the specific saccharide lactose did not affect porphyrin binding significantly, indicating that the sugar and the porphyrin bind at different sites. The Ka values obtained for the binding of different porphyrins with TCSL at 25 degrees C were in the range of 2 x 10(3)-5 x 10(5) m(-1). Association constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (CuTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpyridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature dependence of the association constants, the thermodynamic parameters change in enthalpy (DeltaH degrees ) and change in entropy (DeltaS degrees ) associated with the binding process were estimated. The thermodynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small, suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPyP to TCSL takes place by a single-step process and at 20 degrees C, the association and dissociation rate constants were 1.89 x 10(4) m(-1).s(-1) and 0.29 s(-1), respectively.     

Binding of Porphyrins by the Tumor-Specific Lectin, Jacalin [Jack Fruit (Artocarpus integrifolia) Agglutinin]

Jacalin (Artocarpus integrifolia agglutinin) specifically recognizes thetumor-associated T-antigenic disaccharide structure,Gal13GalNAc. Porphyrins and their derivatives are currently used asphotosensitizers in photodynamic therapy to treat malignant tumors. In thisstudy, the interaction of several free base porphyrins and their metalderivatives with jacalin is investigated by absorption and fluorescencespectroscopy. Each lectin subunit was found to bind one porphyrin moleculeand the association constants were estimated to be in the range of2.4×10³M^–1 to 1.3×10⁵M^–1 at room temperaturefor the interaction of different porphyrins with jacalin. These values arein the same range as those obtained for the interaction of monosaccharidesto jacalin. Both free lectin and lectin saturated with the specificsaccharide were found to bind different porphyrins with comparable bindingstrength indicating that porphyrin binding takes place at a site differentfrom the sugar binding site. Further, both anionic and cationic porphyrinswere found to interact with the lectin with comparable affinity, clearlyindicating that the charge on the porphyrin does not play any role in thebinding process and that most likely the interaction is mediated byhydrophobic forces. These results suggest that jacalin and other lectins maypotentially be useful for targeted delivery of porphyrins to tumor tissuesin photodynamic therapy.     

The Interaction of New Pyridylporphyrins with Bovine Serum Albumin

The interaction of meso-tetra(4-N-hydroxyethylpyridyl)porphyrin, meso-tetra(3-N-hydroxyethylpyridyl)porphyrin, and their zinc complexes with bovine serum albumin (BSA) was studied by electronic spectroscopy, CD, and equilibrium dialysis at pH 7.2. The titration of the porphyrins with BSA was accompanied by a decrease in light absorption and a bathochromic shift of the Soret band, as well as by the appearance of an isobestic point. The porphyrin interaction with BSA also led to the induction of positive CD spectra in the visible region, which is explained by the porphyrin sorption on the protein globule. The equilibrium dialysis helped in determining the stoichiometry of binding and the binding constants of the porphyrins under study with BSA using Scatchard plots. This interaction is nonspecific and reversible.     

Energetics of carbohydrate binding to Momordica charantia (bitter gourd) lectin: an isothermal titration calorimetric study

Physico-chemical and carbohydrate binding studies have been carried out on the Momordica charantia (bitter gourd) seed lectin (MCL). The lectin activity is maximal in the pH range 7.4-11.0, but decreases steeply below pH 7.0. The lectin activity is mostly unaffected in the temperature range 4-50 degrees C, but a sharp decrease is seen between 50 and 60 degrees C, which could be correlated to changes in the structure of the protein as seen by circular dichroism and fluorescence spectroscopy. Isothermal titration calorimetric studies show that the tetrameric MCL binds two sugar molecules and the binding constants (Kb), determined at 288.15 K, for various saccharides were found to vary between 7.3 x 10(3) and 1.52 x 10(4)M(-1). The binding reactions for all the saccharides investigated were essentially enthalpy driven, with the binding enthalpies (DeltaHb) at 288.15 K being in the range of -50.99 and -43.39 kJ mol(-1), whereas the contribution to the binding reaction from the entropy of binding was negative, with values of binding entropy (DeltaSb) ranging between -99.2 and -72.0 J mol(-1)K(-1) at 288.15 K. Changes in heat capacity (DeltaCp) for the binding of disaccharides, lactose and lactulose, were significantly larger in magnitude than those obtained for the monosaccharides, methyl-beta-D-galactopyranoside, and methyl-alpha-D-galactopyranoside, and could be correlated reasonably well with the surface areas of these ligands. Enthalpy-entropy compensation was observed for all the sugars studied, suggesting that water structure plays an important role in the overall binding reaction. CD spectroscopy indicates that carbohydrate binding does not lead to significant changes in the secondary and tertiary structures of MCL, suggesting that the carbohydrate binding sites on this lectin are mostly preformed.     

Haematoporphyrin and OO''-diacetylhaematoporphyrin binding by serum and cellular proteins. Implications for the clearance of these photochemotherapeutic agents by cells.

Haematoporphyrin derivative (HpD), a mixture of porphyrins, is currently used as a photochemotherapeutic agent in the treatment of neoplasias. The interaction of purified components of HpD with serum and cellular proteins was investigated using absorption and fluorescence spectroscopy. The interactions of haematoporphyrin and OO'-diacetylhaematoporphyrin with human albumin and with haemopexin, the two major serum porphyrin-binding proteins, show stoichiometries of 1 mol of porphyrin bound per mol of protein. The apparent dissociation constants, Kd, are in the range of 1-2 microM for albumin and 3-4 microM for haemopexin. These two major components of HpD would, after intravenous injection, bind to albumin and circulate in serum as albumin complexes. Free porphyrin rather than porphyrin bound to albumin interacts with Morris hepatoma tissue culture cells. A rapid high-affinity saturable transport system operates at free porphyrin concentrations of less than 2 microM. In addition, fluorescence spectra show that components in rat liver cytosol can bind haematoporphyrin and OO'-diacetylhaematoporphyrin and distinguish these binders from those present in rat serum.     

Optical absorption and fluorescence spectroscopy studies of ground state melanin-cationic porphyrins complexes.

Optical absorption and fluorescence spectroscopies were employed in the study of the interaction between synthetic L-dopa (dihydroxyphenylalanine) melanin and the cationic porphyrins tetrakis(4-N-methylpyridyl) porphyrin (TMPyP), tetrakis(4-N-benzylpyridyl)porphyrin (TBzPyP), zinc tetrakis(4-N-methylpyridyl)porphyrin (ZnTMPyP) and zinc tetrakis (4-N-benzylpyridyl)porphyrin (ZnTBzPyP). Optical absorption and fluorescence properties of the porphyrins were dependent on the symmetry of the central ring. No evidence was found for dimerization of the porphyrins in phosphate buffer, pH 7, in the concentration range between 4 x 10(-8) to 5 x 10(-5) M. Addition of L-dopa melanin red shifted the optical absorption spectra of porphyrins, concomitant to broadening and reduction in intensity of the bands. L-Dopa melanin also strongly quenched the fluorescence of the porphyrins. Time resolution of the fluorescence decay of porphyrins showed at least two lifetimes that were only slightly modified in the presence of melanin. The interaction between melanin and porphyrin resulted in the formation of non-fluorescent ground state complexes. It was found that there are two different classes of binding sites in melanin for complexation with cationic porphyrins and the values of dissociation constants are of the order of 10(-8) M. These values and the number of binding sites are dependent on the nature of the porphyrins. It was shown that the binding has electrostatic origin, but it is also affected by metal coordination and hydrophobic interaction.     

Characterization of the adenine binding sites of two Dolichos biflorus lectins.

The seed lectin and a stem and leaf lectin (DB58) from Dolichos biflorus have high-affinity hydrophobic sites that bind to adenine. The present study employs a centrifugal filtration assay to characterize these sites. The seed lectin contains two identical sites with Ka's of 7.31 x 10(5) L/mol whereas DB58 has a single site with a Ka of 1.07 x 10(6) L/mol. The relative affinities of these sites for a host of adenine analogs and derivatives were determined by competitive displacement assays. The most effective competitors for adenine were the cytokinins, a class of plant hormone, for which the lectins had apparent Ka's of 1.96 x 10(5)-4.90 x 10(4) L/mol. Direct binding of the cytokinin 6-(benzylamino)purine (BAP) to both lectins showed positive cooperativity for only the seed lectin, indicating the interaction of this ligand with more than one class of hydrophobic binding site. Fluorescence enhancement assays demonstrate cooperativity between hydrophobic sites of the seed lectin and also suggest that BAP binds to more than one class of site.     

Syntheses and DNA binding of new cationic porphyrin-tetrapeptide conjugates

Recently cationic porphyrin-peptide conjugates were synthesized to enhance the cellular uptake of porphyrins or deliver the peptide moiety to the close vicinity of nucleic acids. DNA binding of such compounds was not systematically studied yet.We synthesized two new porphyrin-tetrapeptide conjugates which can be considered as a typical monomer unit corresponding to the branches of porphyrin-polymeric branched chain polypeptide conjugates. Tetra-peptides were linked to the tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrin derivatives, and their peptide conjugates was investigated with comprehensive spectroscopic methods. Titration of porphyrin conjugates with DNA showed changes in Soret bands with bathocromic shifts and hypochromicities. Decomposition of absorption spectra suggested the formation of two populations of bound porphyrins.Evidence provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and induced CD signals reveals that peptide conjugates of di- and tricationic porphyrins bind to DNA by two distinct binding modes which can be identified as intercalation and external binding. Tri-cationic structure and elimination of negative charges in the peptide conjugates are preferable for the binding. Our findings provide essential information for the design of DNA-targeted porphyrin-peptide conjugates.     

Regulation of soluble guanylate cyclase activity by porphyrins and metalloporphyrins

Alterations of the chemical structure of protoporphyrin IX markedly altered the activation of soluble guanylate cyclase purified from bovine lung. Hydrophobic side chains at positions 2 and 4 and vicinal propionic acid residues at positions 6 and 7 of the porphyrin ring (protoporphyrin IX, mesoporphyrin IX) were essential for maximal enzyme activation (Ka = 7-8 nM; Vmax = 6-8 mumol of cGMP/min/mg). Substitution of hydrophobic with polar groups (hematoporphyrin IX, coproporphyrin III), or with hydrogen atoms ( deuteroporphyrin IX), and methylation of propionate residues resulted in decreased enzyme stimulation. Stimulatory porphyrins increased the Vmax and the apparent affinities of enzyme for MgGTP and uncomplexed Mg2+. An open central core in the porphyrin ring was essential for enzyme activation. The pyrrolic nitrogen adduct, N-phenylprotoporphyrin IX, was inhibitory and competitive with protoporphyrin IX (KI = 73 nM). Similarly, metalloporphyrins inhibited enzymatic activity and ferro-protoporphyrin IX (KI = 350 nM), zinc-protoporphyrin IX (KI = 50 nM) and manganese-protoporphyrin IX (KI = 9 nM) were competitive with protoporphyrin IX. Inhibitory porphyrins and metalloporphyrins also prevented enzyme activation by S-nitroso-N- acetylpenicillamine and NO. Guanylate cyclase reconstituted with such porphyrins required higher concentrations of protoporphyrin IX for further activation and were not activated by NO. Thus, porphyrins, metalloporphyrins, and NO appeared to interact at a common binding site on guanylate cyclase. This common site is likely that which normally binds heme and, therefore, NO-heme when the heme-containing enzyme is exposed to NO. Thus, NO and nitroso compounds may react with enzyme-bound heme to generate a modified porphyrin which structurally resembles protoporphyrin IX in its interaction with guanylate cyclase.     

Antibody variable region binding by Staphylococcal protein A: thermodynamic analysis and location of the Fv binding site on E-domain.

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (V(H)), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3-4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5+/-0.5 x 10(5) M(-1). A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0+/-0.1, Ka = 2.0+/-0.3 x 10(5) M(-1), and deltaH = -7.1+/-0.4 kcal mol(-1). Similar binding thermodynamics are obtained for titration of the isolated V(H) domain with E-domain indicating that the E-domain binding site on Fab resides within V(H). E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2+/-0.1, Ka > 1.0 x 10(7) M(-1), and deltaH = -24.6+/-0.6 kcal mol(-1). Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the V(H) domain implicated previously in protein A binding.     

Selective binding of cholesterol by recombinant fatty acid binding proteins

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.     

Interaction of new pyridylporphyrins with bovine serum albumin

The interaction of meso-tetra(4-N-hydroxyethylpyridyl)porphyrin, meso-tetra(3-N-hydroxyethylpyridyl)porphyrin, and their zinc complexes with bovine serum albumin (BSA) was studied by electronic spectroscopy, CD, and equilibrium dialysis at pH 7.2. The titration of the porphyrins with BSA was accompanied by a decrease in light absorption and a bathochromic shift of the Soret band, as well as by the appearance of an isobestic point. The porphyrin interaction with BSA also led to the induction of positive CD spectra in the visible region, which is explained by the porphyrin sorption on the protein globule. The equilibrium dialysis helped in determining the stoichiometry of binding and the binding constants of the porphyrins under study with BSA using Scatchard plots. This interaction is nonspecific and reversible. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous beta-galactoside-binding lectin (galaptin).

Glutaraldehyde-polymerized human splenic galaptin, a beta-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative to native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4- and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4 degrees C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 x 10(7) binding sites/cell with affinity constant Ka = 2.2 x 10(5) M for dimeric galaptin and 17 x 10(7) binding sites/cell with Ka = 3.4 x 10(5) M-1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30-50% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively. Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble ligands.     

Tricationic pyridium porphyrins appending different peripheral substituents: experimental and DFT studies on their interactions with DNA

Four tricationic pyridium porphyrins appending hydroxyphenyl, methoxyphenyl, propionoxyphenyl or carboxyphenyl group at meso-20-position of porphyrin core have been synthesized and their abilities to bind and cleave DNA have been investigated. Using a combination of absorption, fluorescence, circular dichroism (CD) spectra, thermal DNA denaturation as well as viscosity measurements, their binding modes and intrinsic binding constants (K_b) to calf DNA (CT DNA) were comparatively studied and also compared with those of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP). The results suggest that the K_b values of these porphyrins are greatly influenced by the number of positive charges and steric hindrance. Theoretical calculations applying the density functional theory (DFT) have been carried out and explain their DNA-binding properties reasonably. The efficiency of DNA photocleavage by these porphyrins shows high dependence on the values of K_b.     

The binding of porphyrin cytochrome c to yeast cytochrome c peroxidase. A fluorescence study of the number of sites and their sensitivity to salt

Porphyrin c, the iron-free derivative of cytochrome c, is a reasonably good model for cytochrome c binding to cytochrome c peroxidase (CcP). It binds with the same stoichiometry but only one-quarter as tightly as cytochrome c. CcP (resting, FeIII) and CcP X CN can both bind up to two molecules of porphyrin c. The binding of the first porphyrin c is tight (kd = 1 X 10(-9) M, pH 6, ionic strength mu = 0, 4 degrees C) and results in quenching of the porphyrin c fluorescence. The binding is sensitive to ionic strength. The binding of the second porphyrin c is looser (Kd unknown) and does not result in quenching of the porphyrin fluorescence. The binding of porphyrin c to the cyano form and the resting forms of CcP cannot be distinguished by our methods. ES is the first acceptor of electrons from c(II) and can bind at least two molecules of porphyrin c. The binding of the first porphyrin c is extremely tight, results in substantial quenching and is insensitive to ionic strength. The binding of porphyrin c to the loose site (Kd = 2 X 10(-9) M, pH 6, 4 degrees C, mu = 0) results, unlike the resting and cyano forms, in quenching of fluorescence of the second porphyrin c. The binding of the second porphyrin c to ES is sensitive to ionic strength. The calculated distances between porphyrin c and the hemes of CcP(FeIII) and ES are approximately 2.5 nm.     

Interactions of water soluble porphyrins with Z-poly(dG-dC).

The water soluble porphyrin tetrakis(4-N-methylpyridyl)porphine (H2TMpyP) and its copper(II) derivative (CuTMpyP) convert Z-poly(dG-dC) to the B-form. For H2TMpyP, the fraction Z character (fr-Z) is given by fr-Z = 1.0 - 21 rO and for CuTMpyP, fr-Z = .94 - 12 rO where rO identical to [Porphyrin]O/[DNA]O. Neither the manganese(III) derivative of of this porphyrin (MnTMpyP) nor tetrakis(2-N-methylpyridyl)porphine (H2TMpyP-2) is nearly as effective at causing the conversion. The former two porphyrins have been shown to intercalate into B-poly(dG-dC) whereas the latter two porphyrins do not. The kinetics of the Z----B conversion are independent of porphyrin or poly(dG-dC) concentration for 1/rO greater than 6. At smaller values of 1/rO, the conversion rate is greatly increased for H2TMpyP and CuTMpyP. The interaction of these porphyrins with Z-poly(dG-dC) follows simple first order kinetics in this latter concentration range. It is proposed that for small values of 1/rO the sequence of events begins with a porphyrin-unassisted distortion of the Z-duplex (with a rate constant of 0.6 s-1) followed by a rapid uptake of porphyrin in what may be an intercalative mode. The porphyrin thus located in Z-regions brings about rapid conversion to the B-form. Binding of H2TMpyP or CuTMpyP to B-regions of a predominantly Z-strand leads to conversion of Z to B. However, this conversion process is considerably slower than when the porphyrins bind directly to Z-regions.     

Lectin from the Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni

Glycan-binding proteins (lectins) are widely expressed in many invertebrates, although the biosynthesis and functions of the lectins are not well understood. Here we report that Manila clam (Ruditapes philippinarum) synthesizes a lectin termed Manila clam lectin (MCL) upon infection with the protozoan parasite Perkinsus olseni. MCL is synthesized in hemocytes as a approximately 74-kDa precursor and secreted into hemolymph where it is converted to 30- and 34-kDa polypeptides. The synthesis of MCL in hemocytes is stimulated by one or more factors in Perkinsus-infected hemolymph, but not directly by Perkinsus itself. MCL can bind to the surfaces of purified hypnospores and zoospores of the parasite, and this binding is inhibitable by either EDTA or GalNAc. Fluorescent beads coated with purified MCL were actively phagocytosed by hemocytes from the clam. Immunohistochemistry showed that secreted MCL is concentrated within cyst-like structures. To define the glycan binding specificity of MCL we examined its binding to an array of biotinylated glycans. MCL recognizes terminal non-reducing beta-linked GalNAc as expressed within the LacdiNAc motif GalNAcbeta1-4GlcNAcbeta1-R and glycans with terminal, non-reducing beta-linked Gal residues. Our results show that the synthesis of MCL is specifically up-regulated upon parasite infection of the clams and may serve as an opsonin through recognition of terminal GalNAc/Gal residues on the parasites.