Abstract
To identify critical amino acid residues for carbohydrate binding of galectins (soluble β-galactoside-binding lectins found in the animal kingdom), site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992)J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.
Similar content being viewed by others
Abbreviations
- EDTA-PBS:
-
2mm EDTA, 20mm Na-phosphate, pH 7.2, 150mm NaCl
- MEPBS:
-
EDTA-PBS containing 4mm β-mercaptoethanol
- IPTG:
-
isopropyl-β-(d)-thiogalactoside
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Teichberg VI, Silman I, Beitsh DD, Resheff G (1975)Proc Natl Acad Sci USA 72:1383–7.
Hirabayashi J, Kasai K (1993)Glycobiology 3:297–304.
Barondes SH, Castronovo V, Cooper DNW, Cummings RD, Drickamer K, Feizi T, Gitt MA, Hirabayashi J, Hughes RC, Kasai K, Leffler H, Liu F-T, Lotan R, Mercurio AM, Monsigny M, Pilli S, Poirer F, Raz A, Rigby PWJ, Rini JM, Wang JL (1994)Cell 76:597–8.
Hirabayashi J, Satoh M, Kasai K (1992)J Biol Chem 267:15485–90.
Oda Y, Herrmann J, Gitt M, Turck CW, Burlingame AL, Barondes SH, Leffler H (1993)J Biol Chem 268:5929–39.
Pfeifer K, Haasemann M, Gamulin V, Bretting H, Fahrenholz F, Muller WEG (1993)Glycobiology 3:179–84.
Hirabayashi J, Kasai K (1991)J Biol Chem 266:23648–53.
Hirabayashi J, Kasai K (1984)Biochem Biophys Res Commun 122:938–44.
Hirabayashi J, Kawasaki H, Suzuki K, Kasai K (1987)J Biochem 101:987–95.
Hirabayashi J, Kasai K (1988)J Biochem 104:1–4.
Hirabayashi J, Ayaki H, Soma G, Kasai K (1989)Biochim Biophys Acta 1008:85–91.
Abbott WM, Feizi T (1989)Biochem J 259:291–4.
Sharma A, Chemelli R, Allen HJ (1990)Biochemistry 29:5309–14.
Bladier D, Le Caer JP, Joubert R, Caron M, Rossier J (1991)Neurochem Int 18:275–81.
Couroud PO, Cadesentini-Borzocz D, Bringman JS, Griffith J, McGrogan M, Nedwin GE (1989)J Biol Chem 264:1310–16.
Quiocho FA (1986)Annu Rev Biochem 55:287–315.
Weis WI, Drickamer K, Hendrickson WA (1992)Nature 360:127–35.
Ackerman SJ, Corrette SE, Rosenberg HF, Bennett JC, Mastrianni DM, Nicholson-Weller A, Weller PF, Chin DT, Tennen DG (1993)J Immunol 150:456–68.
Ohyama Y, Kasai K (1988)J Biochem 104:173–7.
Gitt MA, Barondes SH (1991)Biochemistry 30:82–9.
Gitt MA, Massa SM, Leffler H, Barondes SH (1992)J Biol Chem 267:10601–6.
Chiariotti L, Wells V, Bruni CB, Mallucci L (1991)Biochim Biophys Acta 1089:54–60.
Gritzmacher CA, Mehl VS, Liu F-T (1992)Biochemistry 31:9533–8.
Hirabayashi J, Kasai K (1993) InLectins and Glycobiology (Gabius H-J, Gabius S eds) pp. 482–91. Berlin: Springer-Verlag.
Hirabayashi J, Ayaki H, Soma G, Kasai K (1989)FEBS Lett 250:161–5.
Hirabayashi J, Sakakura Y, Kasai K (1993) InLectins and Glycobiology (Gabius, H-J, Gabius S eds) pp. 474–81. Berlin: Springer-Verlag.
Harrison FL, Wilson TJG (1992)J Cell Sci 101:635–46.
Hirabayashi J, Kasai K (1992)J Chromatogr 597:181–7.
Lobsanov YD, Gitt MA, Leffler H, Barondes SH, Rini JM (1993)J Biol Chem 268:27034–8.
Hirabayashi J (1993)Trends Glycosci Glycotechnol 5:251–70.
Hirabayashi J (1994) InLectins, Biology, Biochemistry, Clinical Biochemistry, Vol. 10 (Bøog-Hansen TC ed.) Sigma (in press).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hirabayashi, J., Kasai, KI. Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1. Glycoconjugate J 11, 437–442 (1994). https://doi.org/10.1007/BF00731280
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00731280