Glycobiology, 2000, Vol. 10, No. 12 1325-1331
© 2000 Oxford University Press
Description of a monomeric prototype galectin from the lizard Podarcis hispanica
Instituto de Química Física "Rocasolano," Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006 Madrid, Spain, and 2Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velazquez 144, E-28006 Madrid, Spain
Received on May 18, 2000; revised on July 28, 2000; accepted on August 3, 2000.
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Abstract |
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Galectins are a continuously expanding family of ß-galactoside-binding lectins present in a variety of evolutionarily divergent animal species. Here we report, for the first time, that expression of galectins extends to the reptilia lineage of lizards. Up to five lactose-binding proteins were isolated from the lizard Podarcis hispanica by affinity chromatography on asialofetuin-Sepharose. The main component, which is most abundantly expressed in skin, was purified from this tissue and further characterized. Under native conditions the protein behaved as a monomer with a molecular mass of 14,500 Da and an isoelectric point of 6.3. Based on sequence homology of the 58 N-terminal amino acid residues with galectins, and on its demonstrated galactoside-binding activity, this lectin we named LG-14 (from Lizard Galectin and 14 kDa) is classified as a new member of the galectin family. LG-14 falls into and strengthen the still thinly populated category of monomeric prototype galectins.
Key words: galectin/lizard/Podarcis hispanica/reptiles/skin
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Galectins are a family of evolutionarily conserved proteins which are defined by their affinity for poly-N-acetyllactosamine glycoconjugates and sequence similarities in the carbohydrate recognition domain (Hirabayashi and Kasai, 1993
; Ahmed and Vasta, 1994; Barondes et al., 1994; Kasai and Hirabayashi, 1996; Gabius, 1997; Hirabayashi, 1997). They are widely distributed from higher vertebrates to lower invertebrates, including mammals, amphibians, fish, birds, nematodes, and sponges, having even been found in the mushroom Coprinus cinereus (Cooper et al., 1997). Therefore, the galectin gene family must have evolved from the start of multicellular organisms, at least a million of years ago. Although during the last 20 years the structure and carbohydrate-binding properties of many galectins have been well established, their physiological functions have not yet been fully understood. The presence of galectins in so many evolutionarily divergent species suggests that they participate in basic cellular functions. In fact, they are supposed to be involved in a variety of important processes occurring in multicellular organisms, such as cell adhesion, cell growth regulation, immunomodulation, apoptosis, tumor spreading, and metastasis (Hirabayashi and Kasai, 1993; Barondes et al., 1994; Hirabayashi, 1997; Rabinovich, 1999). If galectins were essential for these biological processes, they would be expected to exist in all animal species. Very recently, candidate galectins have been found in the genome of the insects Drosophila and Anopheles, of viruses and of the plant Arabadopsis (Cooper and Barondes, 1999). In this paper, we report on the first reptilian galectin, isolated from the skin of the lizard Podarcis hispanica. |
Results |
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Isolation of galactose-binding lectins from lizards.
Several lactose-binding lectins were isolated from soluble extracts of whole Podarcis hispanica homogenates by affinity chromatography on asialofetuin-Sepharose. A predominant protein band with an apparent molecular mass of 14 kDa in SDS–PAGE was observed (Figure 1, lane 1). In addition, other minor bands with apparent molecular masses of 16, 17, 22, and 35 kDa could be detected. The relative intensity of these bands varied among different lizard preparations. The origin of such differences in the pattern remains to be established, but it could be related to seasonal variations and/or to the impact of storage of lizards at –20°C before processing.
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Western blot analysis using polyclonal antibodies raised against bovine galectin-1 and the two chicken galectins, CG-14 and CG-16, indicated that all these proteins were immunologically related to galectins (Figure 1). The 14 kDa and 16 kDa proteins cross-reacted with the three antibodies used (Figure 1). Overlay with anti-galectin-1 antibody allowed the detection of the 35 kDa protein (Figure 1, lane 2), even when this band was not visible by silver staining. Cross-reaction was also observed for the 17 kDa and 22 kDa proteins with anti-CG-14 antibody (Figure 1, lane 3).
A pool of three lizards was used to study the distribution of these proteins in different tissues. The following fractions were investigated: skin, intestine, liver, sexual organs and kidney, lung and heart, and a body fraction comprising muscle, bone, and nervous tissue. Analysis by SDS–PAGE of the affinity purified proteins (Figure 2) showed that the 14 kDa protein band is abundantly present in the body fraction and specially in the skin. Minor amounts of this protein band are found in most of the tissues. The 16 kDa protein band was found in the skin, intestine and in the body fraction and the 35 kDa protein band was detected only in the body fraction. Two new protein bands with molecular masses between 14 and 16 kDa were observed in liver and in the fraction including heart and lung. No proteins were detected in the fraction composed by sexual organs and kidney (not shown). N-Terminal amino acid sequence analysis (see below) of the 14 kDa protein isolated from the skin and from the body fraction indicates that these tissues most probably express the same galectin. The yield of the 14 kDa protein purified from whole lizard homogenates, as estimated by amino acid composition analysis, was 25–27 µg/g lizard. This galectin, we have named LG-14, was purified from the skin and further characterised.
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Molecular mass and isoelectric point of LG-14
MALDI-TOF mass spectrometry of LG-14 gave a molecular mass of 14,500 Da per monomer. On size exclusion chromatography in the presence of lactose, the lectin eluted as a single peak with an apparent molecular mass of 14 kDa. A single species with an apparent molecular mass between 14 and 17 kDa was observed by equilibrium sedimentation in the absence of lactose and at a protein concentration of 0.5 mg/ml. Although the exact molecular mass of the galectin could not be calculated from this experiment, the result obtained clearly indicated that, under these conditions, the galectin behaved as a monomer. Size exclusion chromatography of the affinity purified proteins from whole lizard homogenates showed that all the 14 kDa proteins eluted at the same position, indicating that the 14 kDa galectin present in the body fraction also behaves as a monomer.
Isoelectric focusing of purified LG-14 from skin gave a single component with a pI of 6.3.
Amino acid composition and N-terminal sequence of LG-14
Amino acid composition is given in Table I. As observed in most prototype galectins, LG-14 contained relatively high amounts of Asx, Glx, and Gly, and a higher content of Phe than Tyr. Four half-Cys were found per molecule. The presence of at least one Trp was deduced from the N-terminal sequence analysis.
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The NH2-terminus of the isolated protein was not blocked and, therefore, partial sequence could be determined by Edman degradation of the nondigested protein (Figure 3). Serendipitously, up to 57 amino acid residues could be identified by N-terminal sequencing. The gap at position 5 (X) most probably corresponds to a Cys residue since, in unmodified proteins, cyst(e)ine residues are degraded during derivatization and, in consequence, they show up as gaps (Henschen, 1986
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The 10 N-terminal amino acid sequence of the 14 kDa protein isolated from the body fraction was identical to that of the 14 kDa protein isolated from the skin.
Comparison of the 58 sequenced N-terminal amino acid residues with the N-terminal sequence of galectins from other species (Figure 3) clearly confirmed that the protein isolated from Podarcis hispanica is a member of the galectin family, the highest homology being exhibited with the prototype galectin-1 (50.88% identity) (Abbott et al., 1989; Hirabayashi and Kasai, 1988), CG-14 (56.14% identity) (Hirabayashi et al., 1987), and CG-16 (49.12% identity) (Sakakura et al., 1990). The percentage of identity decreased for other mammalian galectins (not shown) and for galectins from phylogenetic distant species as nematodes (Hirabayashi et al., 1996), sponge (Pfeifer et al., 1993), and fungi (Cooper et al., 1997) (identities below 30%). Within the sequenced segment, the relevant amino acid residues found to be involved in protein-sugar contacts in the crystal structure of the bovine galectin-1-N-acetyllactosamine complex (Liao et al., 1994), namely His43, Asn45, Arg47, His51, Asp53, and Val58 (according to the LG-14 numeration) are preserved in LG-14.
Sugar binding ability
Radioiodination of purified LG-14 using Bolton-Hunter reagent resulted in an active preparation that bound to asialofibrin in a concentration-dependent manner (Figure 4). About 8% of the 125I-galectin bound to 120 µg asialofibrin films, and 80% of the binding was inhibited by 0.1 M lactose. Elution of the radioactivity bound to the asialofibrin films with buffer containing SDS and ß-mercaptoethanol and subsequent analysis by SDS–PAGE and autoradiography showed the presence of pure galectin. The low percentage of specifically bound 125I-LG-14 was similar to that observed for monomeric chicken CG-14 in the same kind of experiments (Solís et al., 1996). The lectin remained active after storage either at 4°C or –20°C at least for 1 week. Overnight dialysis against PBS resulted in the loss of about 30% of the binding and this was completely restored after incubation for 1 h with ß-mercaptoethanol, thus demonstrating the requirement of a reducing environment for sugar binding activity.
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Galactose, lactose, and N-acetyllactosamine, were tested as inhibitors of the binding of 125I-LG-14 to asialofibrin films. Apparent dissociation constants were calculated from the plot of the reciprocal of the LG-14 fraction bound to the film versus the sugar concentration (Rivera-Sagredo et al., 1991
). As shown in Table II, the affinity of LG-14 for the three sugars was roughly in the range of that previously determined for bovine galectin-1 (Solís et al., 1994), CG-14 and CG-16 (Solís et al., 1996).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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LG-14, the first reptilian galectin described to date, has been isolated from the skin of the lizard Podarchis hispanica. Several other proteins exhibiting asialofetuin-binding ability and immunological cross-reactivity with galectins have also been observed in this and other tissues, strongly suggesting the occurrence of different members of the galectin family in this species. The expression of several galectins in one organism thus appears as a constant feature of this lectin family, common to all animal species in which galectins have been identified so far, including lizards.
The yield of LG-14, the most abundant galectin in Podarchis hispanica (25–27 µg/g of whole lizard), is comparable to that found in other animal species. For example, bovine spleen contains between 35–40 µg /g of wet tissue (Ahmed et al., 1996b) and the skin of the frog Xenopus laevis expresses as much as 0.8 mg/g (Marschal et al., 1992). Also different species of annelida (Hirabayashi et al., 1998) and nematode (Hirabayashi et al., 1996) contain between 17 and 50 µg of galectin per gram of worm.
LG-14, with a molecular mass of 14,500 by mass spectrometry, can be classified as a prototype galectin. Furthermore, comparison of the 58 N-terminal amino acid residues with those of other galectins reveals that LG-14 is closer in sequence to mammalian galectin-1 (Hirabayashi and Kasai, 1988; Abbott et al., 1989) and the chicken lectins CG-14 (Hirabayashi et al., 1987) and CG-16 (Sakakura et al., 1990) than to other proto-type galectins. Although the sequenced fragment of LG-14 does not comprise the complete carbohydrate-binding site, within the sequenced segment all the relevant amino acid residues involved in sugar binding are identical in these four galectins. And accordingly, in broad outline LG-14 exhibits the typical sugar binding ability of galectins such as bovine galectin-1 (Solís et al., 1994) and the chicken galectins (Solís et al., 1996), the affinity of the binding increasing in the order: galactose < lactose < N-acetyllactosamine.
Prototype galectins have been found in all animal species in which galectins have been so far identified, most frequently in the form of noncovalent dimers: mammalian galectins 1 and 2 and most prototype nonmammalian galectins, including chicken CG-16, all exhibit a dimeric quaternary structure in solution, at or below concentrations at which they usually occur in cells and tissues (Hirabayashi, 1997). However, LG-14 behaves as a monomer at a relatively high concentration (0.03 mM) either in the absence or in the presence of the carbohydrate ligand. Such a distinctive feature has been experimentally demonstrated only for the prototype rat galectin-5 (Gitt et al., 1995) and chicken CG-14 (Beyer et al., 1980).
X-Ray crystallography of several dimeric prototype galectins (Lobsanov et al., 1993; Bourne et al., 1994; Liao et al., 1994; Varela et al., 1999) has shown that dimerization derives from extended ß-sheet interactions across the two monomers, with the N- and C-terminus of each monomer at the dimer interface. Several non-polar residues from both monomers extend towards the interior of the molecule and create a hydrophobic core also contributing to the stability of the dimer. The presence in galectin-5 of an extended N-terminal peptide, as compared to other prototype galectins, could be reasonably proposed to underlie the monomeric character of this lectin. Such an extended N-terminus might easily interfere with the above-described packing of the two monomers into the dimer. For LG-14, similarly to chicken CG-14, however, no immediate explanation is found. Structural information on the dimer interface of the homologous chicken galectin CG-16 appears to indicate that the monomer nature of CG-14 is determined by only a few substitutions of key amino acid residues at the dimer interface, while the overall topology of the carbohydrate-recognition domain is preserved (Varela et al., 1999). For example, substitution of Val6 in CG-16 by Cys in CG-14 might affect dimerization due to the reduction of the non-polar character of the interface. Interestingly, a Cys residue is also found at this position in LG-14. In addition, a Phe to Trp substitution at position 133 in CG-14 is expected to compromise the compactness of the dimer because of steric conflict to accommodate tryptophan’s lateral chain (Varela et al., 1999). Sequence information on the C-terminal half of LG-14 will provide additional insights into the specific contact points critically affecting the monomer/dimer nature of these lectins.
The results here reported indicated that LG-14 is present in the body fraction and most abundantly in the skin. This preferential expression recalls that of chicken CG-14, which is abundantly found in keratinized embryonic skin (Oda et al., 1989). On the other hand, mammalian galectin-7, with which LG-14 displays only about 30% N-sequence identity, is present in both keratinized and non-keratinized stratified epithelia (Magnaldo et al., 1998). Keratinization of reptilian skin is very extensive. In lizards, similarly to snakes, shedding of the cornified layer, termed molting or ecdysis, results in removal of extensive sections of superficial epidermis with concomitant inner generation of a new epidermis. Localization of LG-14 within the different epidermal/dermal layers and investigation of the presumptive changes in expression related with molting will surely provide invaluable clues to the possible relation of this monomeric lectin with skin generation and keratinization.
Overall the similarities observed between lizard LG-14 and avian CG-14, including sequence homology, close to neutral isoelectric point, and monomeric structure, put forward the possible existence of similar galectins in the different living clades of diapsids, which include lizards and snakes, crocodiles and birds (Figure 5). Their occurrence in birds is reinforced by the description in quail of a monomeric galactose-binding lectin with a molecular mass of 14,500 and a pI of 6.2 (Fang and Ceri, 1990), although no sequence information is available.
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Amniotes and living amphibians are the two main evolutionary lineages of extant terrestrial vertebrates (Figure 5). Amniotes first diverged into two lines: one line that culminated in living mammals and another line that embraces all the living reptiles (including birds). To date no monomeric galectin of the type found in lizard and birds has been described in either mammals or amphibians. Examination of galectins in turtles and crocodiles will prove whether the monomeric galectins evolved at the time of divergence of mammals and reptiles, whether they are confined to diapsids or whether they are the result of parallel evolution.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Materials
CNBr-activated Sepharose 4B and Superose 12 were from Pharmacia. Dialysis bags were Spectra/Por Membrane MWCO: 6–8,000 (Spectrum, USA). Alkaline phosphatase-conjugated goat anti-rabbit IgG was from Promega Co. (USA). Affinity purified polyclonal IgGs from antisera raised against bovine galectin-1 and against chicken galectins CG-14 and CG-16 obtained as previously described (Gabius et al., 1991
) were kindly provided by Prof. H.-J.Gabius (Ludwig-Maximilians-Universität, München, Germany).
Animals
Healthy adult specimens of the lizard Podarcis hispanica were captured in the surroundings of Burjasot (Valencia, Spain) and maintained in terraria, simulating their natural habitat conditions. They were killed by freezing in liquid N2 and kept at –20°C until processed.
Preparation of galectins
Whole lizards or separated organs and tissues were homogenized using a Sorwall Omni-Mixer in 10 volumes of 5 mM sodium phosphate buffer pH 7.2, 0.15 M NaCl (PBS), containing 5 mM ß-mercaptoethanol, 0.1 M lactose, 60 µM phenylmethylsulfonyl fluoride, 10 µM p-nitrophenyl-p'-guanidinobenzoate, and 30 µg/ml aprotinin. After shaking for 30 min at 4°C, the homogenates were centrifuged at 35,000 x g for 45 min. The sediment was extracted again as before. The supernatants were combined and exhaustively dialyzed against PBS containing 5 mM ß-mercaptoethanol. Precipitated material was removed by centrifugation at 100,000 x g for 1 h and the clear supernatant was applied to a column of asialofetuin-Sepharose (1.5 x 3 cm, 8 mg asialofetuin/ml gel) pre-equilibrated with PBS containing 5 mM ß-mercaptoethanol. The column was washed with 10 bed volumes of the equilibrating buffer, and bound proteins were eluted with 3 bed volumes of the same buffer containing 0.1 M lactose. The bound fraction was concentrated by ultrafiltration using Centriplus 10 and Centricon 10 devices (Amicon, Inc.). All the operations were carried out at 4°C.
Affinity purified extracts were chromatographed on a 2 ml Bio-Scale DEAE Column (Bio-Rad) equilibrated with 10 mM phosphate pH 7.6, containing 5 mM ß-mercaptoethanol. Elution was carried out with the same buffer at a flow rate of 1 ml/min using a Pharmacia Fast Protein Liquid Chromatography system (FPLC). Under these conditions all the proteins ran through the column with different elution times. Fractions containing pure 14 kDa protein, as evidenced by SDS–PAGE analysis, were concentrated and used for further analysis.
Gel filtration chromatography
The chromatography was performed on a Superose 12 column (Pharmacia Biotech. Inc.) equilibrated with PBS containing 5 mM ß-mercaptoethanol and 0.1 M lactose. Elution was carried out at a flow rate of 0.5 ml/min using a Pharmacia FPLC system. Alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), 
-glycerophosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12.4 kDa) were run as standards under the same conditions.
Electrophoretic methods
SDS–polyacrylamide gel electrophoresis under reducing conditions was performed on 15% polyacrylamide separating gels using the Laemmli system (Laemmli, 1970). Isoelectric focusing was carried out in pH 3–10 gels using a MultiTemp II system (Pharmacia Biotech). The pH gradient was determined on 5 mm gel slides eluted with 1 ml of distilled water. After electrophoresis and IEF, proteins were detected by silver staining (Morrisey, 1981).
For Western blotting, proteins separated by SDS–PAGE were electrotransferred to Immobilon-Psq (Bio-Rad) using a semidry blotting system (Pharmacia) as described by Towbin et al. (1979). Membranes were blocked with 3% bovine serum albumin in 10 mM Tris–HCl pH 7.8 containing 0.15 M NaCl (TBS), incubated with the different rabbit anti-galectin IgGs (final concentration 0.2–2 µg/ml) at room temperature for 2 h, washed with TBS, and then incubated for 1 h with alkaline phosphatase-conjugated goat anti-rabbit antiserum (diluted 1:6,000). Visualization of positive reaction was carried out by staining with 5-bromo-4-chloro-3'-indolyl phosphate and nitro blue tetrazolium (Boehringer Mannheim) as specified by the manufacturers.
Amino acid composition
Protein sample was subjected to vapor-phase hydrolysis with 6 M constant boiling HCl (Pierce Chemical Co.) for 24 h at 110°C in vacuum. The hydrolyzate was analyzed in a Pharmacia Biochrom 20 amino acid analyzer. Cysteine was determined as cysteic acid by oxidation of the protein with formic acid prior to hydrolysis, according to Hirs (1967).
N-Terminal amino acid sequence analysis
A sample of pure LG-14 from the skin was loaded on a precycled Biobrene Plus-coated glass filter. Affinity purified proteins from body fraction homogenates were transferred, after SDS–PAGE, to Immobilonsq. N-terminal amino acid sequence analysis was carried out in a Perkin Elmer/Applied Biosystems Procise 494 microsequencer running in pulsed-liquid mode.
MALDI-TOF mass spectrometry
A saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid in aqueous 33% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix. The sample was mixed with an equal volume of matrix solution. One microliter of this mixture was deposited on the metal target, allowed to air-dry and introduced into the mass spectrometer. The mass spectrum was recorded with a MALDI-TOF mass spectrometer (Bruker model Biflex). The spectrum was obtained by summing up 80 laser shots.
Sedimentation equilibrium
The experiments were carried out by centrifugation of a 70 µl sample of the lectin in PBS (0.5 mg/ml, according to amino acid analysis) at 25°C and 20,000 r.p.m. in an Optima XL-A analytical ultracentrifuge (Beckman Instruments). Radial scans were taken at 4 h intervals at 280 nm until the equilibrium condition was reached. Base-line offsets were determined taking a radial after running the sample for 5 h at 42,000 r.p.m.. Conservation of mass in the cell was checked during all the experiment. Apparent molecular mass was obtained by fitting the data to a sedimentation equilibrium model for single species, using the signal conservation algorithm from EQASSOC program (Milton, 1994). The partial specific volume calculated from amino acid composition was 0.743 ml g–1 at 25°C (Laue et al., 1992).
Sugar binding assays
Previous to radioiodination, pure 14 kDa protein was dialyzed (4h at 4°C) against 0.1 M borate pH 8.4 containing 0.1 M lactose to remove ß-mercaptoethanol and radioiodinated in the presence of 0.1 M lactose using Bolton-Hunter reagent (Amersham Int.) according to the manufacturer’s recommendations. The labeled lectin was separated from excess reagent by gel filtration chromatography on a PD 10 column (Pharmacia) equilibrated with PBS containing 2 mM ß-mercaptoethanol. Binding of the labeled lectin was assayed using asialofibrin films immobilized on the surface of plastic microwells. Asialofibrin films were prepared from a 3 mg/ml asialofibrinogen solution (40 µl/well) in PBS by addition of 2 U/mg asialofibrinogen of bovine thrombin (Sigma) and incubation for 1 h at 25°C. Plates were air-dried, washed with PBS, and blocked with 3% bovine serum albumin. Wells were incubated for 2 h at 25°C with 50 µl of the 125I-galectin solution (about 15,000 c.p.m.) in PBS containing 2 mM ß-mercaptoethanol. The affinity of the lectin for the different sugars was estimated by determining the amount of 125I-galectin bound to the asialofibrin films in the presence of different concentrations of the sugar (from 0.1 to 8 mM; up to 80 mM for galactose).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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We are grateful to Dr. Carlos García López (Universidad de Valencia, Spain) for providing the lizard specimens, to Prof. Hans-Joachim Gabius (Ludwig-Maximilians-Universität, München, Germany) for generous gift of anti-galectin antibodies, to Dr. Germán Rivas (Centro de Investigaciones Biológicas, C.S.I.C., Madrid, Spain) for analytical ultracentrifugation studies, to Maria Pernas (Universidad de Vigo, Spain) for isoelectric focussing experiments and to Dr. Manuel Nieto-Díaz for his valuable advice and helpful discussions on phylogeny. This work was supported by a grant from the Dirección General de Investigación Científica y Técnica (Grant PB98–0650).
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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PBS, phosphate-buffered saline; TBS, Tris-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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