Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis

Galectins are a family of b-galactoside-binding lectins that onsynthesis are either translocated into the nucleus or releasedto the extracellular space. Their developmentally regulatedexpression, extracellular location, and affinity for extracel-lular components (such as laminin and fibronectin) suggest arole in embryonic development, but so far this has not beenunequivocally established. Zebrafish constitute an ideal modelfor developmental studies because of their external fertiliza-tion, transparent embryos, rapid growth, and availability of alarge collection of mutants. As a first step in addressing thebiological roles in zebrafish embryogenesis, we identified andcharacterizedmembersofthethreegalectintypes:threeproto-galectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimeragalectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 pre-ferentially binds to N-acetyllactosamine. Genomic structureof Drgal1-L2 revealed four exons, with the exon–intronboundaries conserved with the mammalian galectin-1.Interestingly, this gene also encodes an alternatively splicedform of Drgal1-L2 that lacks eight amino acids near thecarbohydrate-binding domain. Zebrafish galectins exhibiteddistinct patterns of temporal expression during embryo devel-opment. Drgal1-L2 is expressed postbud stage, and itsexpression is strikingly specific to the notochord. In contrast,Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3,Drgal3, and Drgal9-L1 are expressed both maternally andzygotically, ubiquitously in the adult tissues. The distincttemporal and spatial patterns of expression of members ofthe zebrafish galectin repertoire suggest that each may playdistinct biological roles during early embryogenesis.Key words: Danio rerio/developmental expression/notochord/specificity/zebrafish galectinsIntroductionThere is growing evidence that complex carbohydrate struc-tures encode information that modulate interactionsbetween cells or between cells and the extracellular matrixby specifically binding to cell surface–associated or solublecarbohydrate-binding receptors (Bakkers et al., 1997;Hathaway and Shur, 1997; Laine, 1997). The expressionpatterns of such carbohydrate-binding receptors are ofparticular interest in the context of development becausethe structures of potential ligand molecules, such as glyco-proteins, glycolipids, and other glycans, are subject tochange by glycosidases and glycosyltransferases (Shur,1993; Wassarman, 1995). The rapidity and complexity ofthe changes in exposed carbohydrate residues during earlydevelopment suggest that they may play important rolesduring embryogenesis.Galectins, a family of b-galactoside-binding development-ally regulated proteins formerly known as S-type lectins,have been proposed to participate in a variety of biologi-cal functions, including cell–cell and cell–extracellularmatrix interactions that mediate developmental processes(Barondes et al., 1994; Hirabayashi, 1997). However, thedetailed mechanisms of their biological role(s) remainunclear. Unlike other lectin families, such as C-type lectins,galectins are a relatively homogeneous, evolutionarily con-served and ubiquitous group, with representatives identifiedin most animal taxa examined so far, including the parazoaand both protostome and deuterostome lineages (Cooper,2002; Hirabayashi, 1997; Vasta et al., 1999). Furthermore,from the biochemical, structural, and genetic standpoints,galectins constitute one of the best-characterized lectinfamilies (Ahmed et al., 1996a; Cho and Cummings, 1995;Liao et al., 1994; Ohyama and Kasai, 1988; Schwarz et al.,1998; Sparrow et al., 1987).Based on structural features, galectins have been classi-fied in three types: proto, chimera, and tandem-repeat(Hirabayashi and Kasai, 1993). Prototype galectins containone carbohydrate-recognition domain (CRD) per subunitand are usually homodimers of noncovalently linkedsubunits. In contrast, chimera-type galectins are monomericwith a C-terminal CRD similar to the proto type, joined toan N-terminal peptide of yet unknown functional proper-ties. Tandem-repeat galectins, in which two CRDs arejoined by a linker peptide, are also monomeric. Proto- andtandem-repeat types make up several distinct galectinsubtypes. Galectin subtypes have been numbered followingtheorderoftheirdiscovery(Barondesetal.,1994),andsofar,14 have been described in mammals (Dunphy et al., 2002).Substantial efforts have been invested toward the elucida-tion of the role(s) of galectins in embryogenesis and devel-opment using mammalian models (Colnot et al., 1997), butbecause of the diversity of the galectin repertoire and thetechnical difficulties in working ex utero with mammalianembryos, results have been inconclusive (Colnot et al.,1998). Lower vertebrates and invertebrates appear to have