Novel Galactose-binding Proteins in Annelida

Abstract

Novel type lectins were found in the phylum Annelida, i.e. in the earthworm, tubifex, leech, and lugworm. The lectins (29–31 kDa) were extracted from the worms without the use of detergent and purified by affinity chromatography on asialofetuin-agarose. On the basis of the partial primary structures of the earthworm Lumbricus terrestris 29-kDa lectin (EW29), degenerate primers were synthesized for use in the reverse transcriptase-polymerase chain reaction. An amplified 155-base pair fragment was used to screen a cDNA library. Four types of full-length clones were obtained, all of which encoded 260 amino acids, but which were found to differ at 29 nucleotide positions. Since three of them resulted in non-silent substitutions, EW29 mRNA was considered to be a mixture of at least three distinct polynucleotides encoding the following proteins: Ala44-Gln197-Ile213 (clone 5), Gly44-Gln197-Val213 (clone 7), and Ala44-His197-Ile213 (clones 8 and 9; different at the nucleotide level, but encoding an identical polypeptide). Genomic polymerase chain reaction using DNA from a single worm revealed that the single worm already had four sets of cDNAs. The EW29 protein showed two features. First, the lectin was composed of two homologous domains (14,500 Da) showing 27% identity with each other. When each of the domains was separately expressed inEscherichia coli, the C-terminal domain was found to bind to asialofetuin-agarose as strongly as the whole protein, whereas the N-terminal domain did not bind and only retardation was observed. EW29 was found to exist as a monomer under non-denaturing conditions. It had significant hemagglutinating activity, which was inhibited by a wide range of galactose-containing saccharides. Second, EW29 contained multiple short conserved motifs, “Gly-X-X-X-Gln-X-Trp.” Similar motifs have been found in many carbohydrate-recognizing proteins from an extensive variety of organisms, e.g. plant lectin ricin B-chain and Clostridium botulinum 33-kDa hemagglutinin. Therefore, these carbohydrate-recognition proteins appear to form a protein superfamily. Novel type lectins were found in the phylum Annelida, i.e. in the earthworm, tubifex, leech, and lugworm. The lectins (29–31 kDa) were extracted from the worms without the use of detergent and purified by affinity chromatography on asialofetuin-agarose. On the basis of the partial primary structures of the earthworm Lumbricus terrestris 29-kDa lectin (EW29), degenerate primers were synthesized for use in the reverse transcriptase-polymerase chain reaction. An amplified 155-base pair fragment was used to screen a cDNA library. Four types of full-length clones were obtained, all of which encoded 260 amino acids, but which were found to differ at 29 nucleotide positions. Since three of them resulted in non-silent substitutions, EW29 mRNA was considered to be a mixture of at least three distinct polynucleotides encoding the following proteins: Ala44-Gln197-Ile213 (clone 5), Gly44-Gln197-Val213 (clone 7), and Ala44-His197-Ile213 (clones 8 and 9; different at the nucleotide level, but encoding an identical polypeptide). Genomic polymerase chain reaction using DNA from a single worm revealed that the single worm already had four sets of cDNAs. The EW29 protein showed two features. First, the lectin was composed of two homologous domains (14,500 Da) showing 27% identity with each other. When each of the domains was separately expressed inEscherichia coli, the C-terminal domain was found to bind to asialofetuin-agarose as strongly as the whole protein, whereas the N-terminal domain did not bind and only retardation was observed. EW29 was found to exist as a monomer under non-denaturing conditions. It had significant hemagglutinating activity, which was inhibited by a wide range of galactose-containing saccharides. Second, EW29 contained multiple short conserved motifs, “Gly-X-X-X-Gln-X-Trp.” Similar motifs have been found in many carbohydrate-recognizing proteins from an extensive variety of organisms, e.g. plant lectin ricin B-chain and Clostridium botulinum 33-kDa hemagglutinin. Therefore, these carbohydrate-recognition proteins appear to form a protein superfamily. Galactose-binding proteins (lectins) represent a distinguished group among lectins because statistics have shown that more than 60% of the lectins reported thus far are galactose-specific (1Doyle R.J. Doyle R.J. Slifkin M. Lectin-Microorganism Interactions. Marcel Dekker, Inc., New York1994: 1-65Google Scholar). A possible explanation for such a preference is that galactose was selected as an important “recognition” saccharide especially in higher organisms. In fact, galactose is used more frequently in higher organisms like mammals, whereas glucose and mannose are recognized by microorganisms such as bacteria and fungi. From a glycochemical viewpoint, galactose has a nature inherently distinct from that of glucose, mannose, or fructose. The latter three monosaccharides are related by “Lobry de Bruyn-Alberta van Ekenstein transformation” (2Speck Jr., J.G. Adv. Carbohydr. Chem. 1958; 13: 63-103Crossref PubMed Scopus (258) Google Scholar). It is well established that in N-linked oligosacharide biosynthesis galactose is incorporated at later stages after removal of glucosyl and mannosyl residues from the common precursor Glc3Man9GlcNAc2 (3Moremane K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (322) Google Scholar, 4Trimble R.B. Verostek M.F. Trends Glycosci. Glycotechnol. 1995; 7: 1-30Crossref Scopus (17) Google Scholar). These observations led one of the authors to present a hypothesis on the origin of elementary hexoses that galactose is a “latecomer” saccharide relative to glucose and mannose (5Hirabayashi J. Q. Rev. Biol. 1996; 71: 365-380Crossref PubMed Scopus (46) Google Scholar). Galactose is exposed at outermost spaces of cells unless it is masked by sialic acids, so that it is easily recognized by various communication molecules by homophilic carbohydrate-carbohydrate (6Hakomori S. Histochem. J. 1992; 24: 771-776Crossref PubMed Scopus (107) Google Scholar) or heterophilic carbohydrate-protein interactions (7Kasai K. Hirabayashi J. J. Biochem. (Tokyo). 1996; 119: 1-8Crossref PubMed Scopus (451) Google Scholar). Among galactose-specific lectins, galectins are unique in that all of the members belonging to this family are galactose-specific (8Barondes S.H. Cooper D.N.W. Gitt M. Leffler H. J. Biol. Chem. 1994; 269: 20807-20810Abstract Full Text PDF PubMed Google Scholar, 9Hirabayashi J. Trends Glycosci. Glycotechnol. 1997; 9Google Scholar). In general, they are soluble and require no metal ion for their activity. Galectins bind most preferentially to lactosamine-containing saccharides of both glycoproteins and glycolipids. Although galectins had long been believed to occur in vertebrates only, they proved to be distributed in much lower organisms as well (10Hirabayashi J. Satoh M. Kasai K. J. Biol. Chem. 1992; 267: 15485-15490Abstract Full Text PDF PubMed Google Scholar, 11Klion A.D. Donelson J.E. Mol. Biochem. Parasitol. 1994; 65: 305-315Crossref PubMed Scopus (50) Google Scholar, 12Hirabayashi J. Ubukata T. Kasai K. J. Biol. Chem. 1996; 271: 2497-2505Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 13Newton S.E. Monti J.R. Greenhalgh C.J. Ashman K. Meeusen E.N.T. Mol. Biochem. Parasitol. 1997; 86: 143-153Crossref PubMed Scopus (45) Google Scholar, 14Pfeifer K. Haasemann M. Gamulin V. Bretting H. Fahrenholz F. Muller W.E.G. Glycobiology. 1993; 3: 179-184Crossref PubMed Scopus (176) Google Scholar, 15Cooper D.N.W. Boulianne R.P. Charlton S. Farrell E.M. Sucher A. Lu B.C. J. Biol. Chem. 1997; 272: 1514-1521Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Although the biological roles of galectins are not fully understood, they are supposed to play multiple roles basic to multicellular organisms, such as in development, differentiation, tumorigenesis, metastasis, apoptosis, etc. In this regard, if galectins are essential for these biological processes, they would be expected to exist in all animal species. However, there have been only few reports of galectins other than in two animal phyla representing deuterostomes and i.e. and On the other if this is not the a that other lectins for the of galectins in such animal have of proteins from the phylum Annelida, by the as that for i.e. in the of detergent and metal and affinity chromatography on asialofetuin-agarose. a lectins were purified from four as earthworm, belonging to the and These proteins with one and had as galectins with to and metal cDNA was for 29-kDa lectin from the earthworm Lumbricus a the earthworm 29-kDa lectin used earthworm 29-kDa polymerase chain carbohydrate-recognition used earthworm 29-kDa polymerase chain carbohydrate-recognition proved to be a which of two homologous domains to it showed no to the However, it showed to proteins such as ricin botulinum etc. It contained conserved multiple of short and were from were from in The worms were with and for lectin were extracted from worms by the for galectins J. Kasai K. Biochem. PubMed Scopus Google Scholar, J. H. K. Kasai K. J. Biochem. (Tokyo). PubMed Scopus Google worms were by in a with of the of soluble proteins had been by the lectins were extracted from the with by for at as the was to and to a of asialofetuin-agarose to A. S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the with protein was with the was by use of the were to under unless was with a The kDa) of the affinity purified lectin from was purified by chromatography on a The was and with both in the and of at a of was by at the was by extensive the by S.H. Scopus Google Scholar) was of was in each well of a with of of in and of the had at for or was of the of various and of lectin that and of saccharides were used in of and that under the were as The of various saccharides on lectin was as J. Ubukata T. Kasai K. J. Biol. Chem. 1996; 271: 2497-2505Abstract Full Text Full Text PDF PubMed Scopus (65) Google earthworm lectin was extracted from of to of the in the of a of various the were to by on a The lectin was by a using lectin and were used at a or from was were in affinity purified lectin or affinity and EW29 by the at with of lectin with of the was by both and The were at in the of purified lectins from earthworm, tubifex, and were purified by chromatography on a was by a of in The lectin were in and were by chromatography on a and were by a protein as J. H. K. Kasai K. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). The were and so in the of In in the of earthworm 29-kDa was to the were Genomic DNA was from two earthworm A. by a J. T. A Scholar). A few worms were used for the from A. and a single worm was used for that from terrestris to the A DNA was by of the polymerase chain reaction of a of earthworm lectin four sets of primers were synthesized on the and as and for was by use of all of the and reverse primers and a for Genomic DNA from the earthworm A. was used as a The amplified DNA with primers and was on the basis of to the and the nucleotide was with an DNA of of galactose-specific lectins from four on is is asialofetuin-agarose. in a primers used in this primers used for primers used for and in are nucleotide to in and for in a in are nucleotide to in and for of full-length a cDNA by was with the fragment with by use of an A was for as by J. T. A Scholar). were and by an in to the The were and to the and to using an for EW29 protein N-terminal domain and C-terminal domain were by a for the of a pair of primers were i.e. and and a full-length fragment encoding 260 amino was amplified with a full-length (clone used as a The fragment was in with and the was used to clones were selected by using EW29 and as J. Kasai K. and Glycobiology. Google Scholar). The was to in of in the of at The protein was by to a of and the for at the The cells were and by and protein was to as (10Hirabayashi J. Satoh M. Kasai K. J. Biol. Chem. 1992; 267: 15485-15490Abstract Full Text PDF PubMed Google Scholar). of for of domains and of primers for an were i.e. of and sets and were other were the as for In to a for the found in the earthworm lectin under DNA was from a single and was with primers and to a full-length of the and of clones were selected and their nucleotide were with one and to from the cDNA on the that galectins are to but are with a the to lectins as for galectins (10Hirabayashi J. Satoh M. Kasai K. J. Biol. Chem. 1992; 267: 15485-15490Abstract Full Text PDF PubMed Google J. Kasai K. Biochem. PubMed Scopus Google Scholar, J. H. K. Kasai K. J. Biochem. (Tokyo). PubMed Scopus Google Scholar) as galactose-specific in the of metal and affinity chromatography on asialofetuin-agarose. A of affinity chromatography on asialofetuin-agarose is shown for earthworm lectin in and the of for all four i.e. earthworm, tubifex, and are shown in after from the were the 29 and The were to reported for kDa) Glycobiology. 1994; 4: PubMed Scopus Google Scholar) or galectins J. Satoh M. Kasai K. J. Biol. Chem. 1992; 267: 15485-15490Abstract Full Text PDF PubMed Google Scholar, 11Klion A.D. Donelson J.E. Mol. Biochem. Parasitol. 1994; 65: 305-315Crossref PubMed Scopus (50) Google Scholar, S.E. Monti J.R. Greenhalgh C.J. Ashman K. Meeusen E.N.T. Mol. Biochem. Parasitol. 1997; 86: 143-153Crossref PubMed Scopus (45) Google Scholar). Since these lectins were in the of metal they were considered not to be The that no detergent was for the the that the lectins are The of purified lectins from the four are in (29–31 earthworm, tubifex, and showed the of multiple of However, the following observations that these were by of the species. When of earthworm lectin was as as possible and was for the of the at a the of the in was not Among the four of the of was in the most The of were of of the (29–31 the the earthworm protein by chromatography and with of Since both identical not at least of the were considered to be of the to at the domains In to the the lectins, by using lectin shown in all of the lectins purified from the other were with the The of with and proteins was and earthworm belonging to the When lectin were used of purified only (29–31 kDa) were observed. is with the that were more affinity purified lectins from earthworm, tubifex, and were purified by chromatography on a not and with The were on a and were by a protein earthworm and to The of in strongly that these lectins are In most of the lectins had multiple by “Gly-X-X-X-Gln-X-Trp.” of the this short showed in of earthworm these and conserved residues that the lectin contained a significant of of by of contained a mixture of two as a contained a mixture of two as a as a as a contained a mixture of two contained a mixture of two contained a mixture of two contained a mixture of two on the amino residues of most earthworm are the as represent amino for which is by after EW29 was and conserved three amino residues in the or residues nucleotide used as a These contained a mixture of two in a on the amino residues of most earthworm are the as represent amino for which is by after EW29 was and conserved three amino residues in the or residues nucleotide used as a Since it that lectins are were on the earthworm 29-kDa lectin (EW29), because was the among the First, various saccharides were for their to the lectin from the earthworm as under a among various only and were found to be On the other glucose, and had no at the used not the of the saccharides were and and the of extracted lectin were and showed the to the lectin at and whereas galactose showed It is that EW29 was with not only but galactose and a of a has been for was to the of EW29 under non-denaturing conditions. The affinity purified earthworm lectin mixture of 29-kDa and was to a in the of in the protein was showing were to of the 29-kDa lectin was considered to exist as a monomer showed that the purified was and of the species. used this as purified EW29 for the of and for the following purified EW29 at the of in the that EW29 has more than one 29-kDa as it was proved to exist as a A of saccharides was for as under saccharides galactose showed is with the of lectin as On the other galactose-containing saccharides showed on which were expressed as in The are as galactose had as and but were than i.e. Among showed the as but their and as well as were was for However, the was the among of at galactose or glucose the by of purified earthworm 29-kDa lectin in in a The was common to galectins in but distinct in (8Barondes S.H. Cooper D.N.W. Gitt M. Leffler H. J. Biol. Chem. 1994; 269: 20807-20810Abstract Full Text PDF PubMed Google Scholar). were in showing affinity to higher affinity to both and and the affinity to among However, EW29 showed for and whereas galectins have such a EW29 showed a preference for such as and to which galectins in not However, the latter is well recognized by J. Gitt S.H. Leffler H. J. Biol. Chem. 1994; Full Text PDF Google Scholar) and EW29 is galactose-specific but to much preference for galactose-containing saccharides than on the of EW29 and four of degenerate primers were synthesized their was by using all of these and reverse and DNA was from the earthworm as a A significant was only the of and was used not The amplified fragment was and the nucleotide was The amino was found to the of as a with no in and the of in the fragment is as a to the fragment was used as a to screen a cDNA of the earthworm by T. a of 8 clones were clones and and their were following as under found the and were of full-length all of which of and and contained identical but their were different clones and with at is identical to but at two i.e. (clone both encoding and (clone (clone encoding The clones and were from at and in the to and of these were only single nucleotide substitutions, and no or was observed. and amino of as a is in The of clones and are with and of the clones were found at the of of if they at the of them were to be (clones and (clones and encoding (clones and (clone encoding and (clone (clones and encoding On the other non-silent were found at only i.e. (clones and (clone for (clones and (clone for and (clones and (clones 8 and for The amino were The three encoded by these four types of cDNA clones were as by 5), Gly44-Gln197-Val213 (clone 7), and Ala44-His197-Ile213 clones 8 and for these were and The well with by The nucleotide did not for or or for N-linked These of The that of the cDNA clones and were identical that the is more than the for the (clones and of nucleotide found in cDNA and clones for earthworm 29-kDa relative to are for to amino in a relative to are for to amino be First, the lectin had no to galectins many metal basic saccharide of and Second, the lectin of two homologous domains kDa) 27% amino identity in under was the of the 29-kDa However, was more the which the In such amino as as the at the or level, using DNA from a single worm of The was amplified by using synthesized and and the were after a all of the clones proved to be to one of the four types of cDNA clones as and clones were to cDNA and that the already at the that the were from for 29-kDa one of the (clone for an in a protein with was and purified by affinity chromatography on asialofetuin-agarose However, many kDa) were as in the of proteins Since these were to bind to the affinity they were considered to be N-terminal or C-terminal domains that after the that had been to asialofetuin-agarose to a by and in and them to N-terminal that the and were C-terminal from and Since all these were in the N-terminal and C-terminal this that only the C-terminal domain activity. On the other the did not significant amino a of protein was It be to N-terminal to form because many i.e. and are in the for both N-terminal and C-terminal domains of and were synthesized The and were expressed in coli, and their were The C-terminal domain to the as as the whole whereas the N-terminal domain did not bind not However, in the latter from the asialofetuin-agarose was Therefore, that the C-terminal domain has than the N-terminal as far as asialofetuin-agarose was the latter When for of multiple protein with a were on after by asialofetuin-agarose However, this only the in the of not that the were by from the proteins in the from other i.e. earthworm and the of this and J. 1994; PubMed Scopus Google Scholar, J. 1994; PubMed Scopus Google Scholar) reported the of galactose-specific lectins, and from the and to have to of the earthworm lectin in the present i.e. in of metal for and for saccharides. they used detergent to lectins, it have to which the lectins were reported that and showed than of the lectins to asialofetuin-agarose was inhibited by and as in the of whereas that of the latter was not inhibited by these saccharides. Since the reported kDa) of was to of lectins (29–31 and purified EW29 with and to be a of the earthworm Galactose-binding lectins in and belonging to T. Biochem. 1997; Scopus (17) Google Scholar) as well to T. Biol. Google Scholar) represent molecules in these Although these lectins were by the as used for partial that they are not related to galectins but form protein family the In fact, the earthworm lectin be extracted with both galactose and as well as whereas galectins in have only affinity to the saccharides. Although to the for lectin it proved to be because removal did not the or of the earthworm The of cDNA of EW29 was in of the lectin proved not to be a of the to the lectins were to be members of a family that many other carbohydrate-recognition least two of proteins have been shown to the multiple short The group of kDa) EW29 in this and 33-kDa of from botulinum type K. K. T. T. K. PubMed Google Scholar) as well as the B-chain of a of plant A of plant of and B-chain have been e.g. ricin J. Biochem. PubMed Scopus Google J. Biol. Chem. Full Text PDF PubMed Google Scholar) from and their related in S. H. K. H. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google and M. H. T. Biochem. 1997; PubMed Scopus Google Scholar). M. K. M. J. Biol. Chem. Full Text PDF PubMed Google M. J. Biochem. PubMed Scopus (50) Google and H. J. S. V. H. S. 1992; Google Scholar) are this type of found in other The group of various J. Biol. Chem. Full Text PDF PubMed Google Scholar) and A F. M. PubMed Scopus Google and H. T. M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and of is a T. T. K. T. S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). all members of the lectin group are to be for for S. Since S. to sialic that is to galactose it be a from an galactose-specific of these members EW29 of two homologous domains both of which as far as that of is for of lectin On the other are not for most of the a the of has been shown to have mannose H. T. M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The is that EW29 as a like ricin and because only the 29-kDa was the has been for ricin with J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the ricin B-chain has two to the two homologous residues the are as and for N-terminal and and as in 8 These residues are to the of the N-terminal and to the one of the C-terminal of residues at the of the is because such residues are frequently found in carbohydrate-recognition of various in galactose-specific lectins Rev. 1995; 24: PubMed Scopus Google A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Although amino these carbohydrate-recognition proteins were it is that these had in their all of the of three each of amino kDa) and motifs in the latter positions. Since the with a M. PubMed Scopus Google Scholar, PubMed Scopus Google it is possible to that the kDa) was the of of this or which is to have had activity. to this one group of the have as kDa) by by of the whereas the other group have to by with have been e.g. for to cells by as has been for H. T. M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). this or lectin for ricin and to be as the for this superfamily. The present on cDNA showed that EW29 is encoded by multiple i.e. The 8 cDNA and clones for cDNA which from the cDNA type (clones and at only two positions. Therefore, the of EW29 in a single worm would not to far four the four types by cDNA clones 8 and 9; or clones and among the four types of the whole at 29 most of them were for the non-silent substitutions, they were homologous and the three would not be expected to different their or be In this regard, three by in the represent these three On the other the that of the cDNA clones were identical to that the is more strongly expressed than other to cDNA clones and are to these The of the lectins is not However, on the that is in in and J. 1996; PubMed Scopus Google Scholar) that the lectin has in the in the of this the it is not the earthworm and other have galectins or However, if they such galectins would not to be with to the In this the hypothesis under the has that of galectins in phyla be by but distinct types of it is of to that no to the lectins have been reported so far in vertebrates and in which galectins are galectins exist in all animal but be extracted with saccharides such as as has been in galectins Hirabayashi J. Kasai K. H. Histochem. J. 1996; PubMed Scopus Google Scholar, J. Biol. 1992; 119: PubMed Scopus Google Scholar). In the present has shown the of lectins in the phylum of the of proteins in multicellular organisms and thus for of T. for with a cDNA for the and for their and Hirabayashi are for their in the worm