Mutational tuning of galectin-3 specificity and biological function.
Galectins are defined by a conserved β-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural β-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic β-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for β-galactosides substituted with GlcNAcβ1-3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.
Salomonsson
Emma
E
Section MIG, Department of Laboratory Medicine, Lund University, 223 62 Lund, Sweden.
Carlsson
Michael C
MC
Osla
Veronica
V
Hendus-Altenburger
Ruth
R
Kahl-Knutson
Barbro
B
Oberg
Christopher T
CT
Sundin
Anders
A
Nilsson
Rickard
R
Nordberg-Karlsson
Eva
E
Nilsson
Ulf J
UJ
Karlsson
Anna
A
Rini
James M
JM
Leffler
Hakon
H
D019943:Amino Acid Substitution; D000818:Animals; D005697:Galactosides / Q000235:genetics / Q000378:metabolism*; D037502:Galectin 3 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D018375:Neutrophil Activation / Q000187:drug effects / Q000502:physiology*; D009504:Neutrophils / Q000378:metabolism*; D013379:Substrate Specificity; D014982:Xenopus laevis
D005697:Galactosides; D037502:Galectin 3; C485095:Lgals3 protein, mouse
J Mol Biol. 2009 Apr 3;387(3):694-705
19361423
Mol Cancer Res. 2009 Oct;7(10):1655-62
19825987
Cancer Res. 2005 Feb 15;65(4):1136-40
15734994
Glycobiology. 2007 Jun;17(6):663-76
17339281
J Biol Chem. 1985 Jan 25;260(2):1067-82
3881421
Glycobiology. 2008 Nov;18(11):905-12
18725453
Chemistry. 2008;14(14):4233-45
18366047
Eur J Biochem. 1998 Jan 15;251(1-2):288-94
9492296
J Biol Chem. 1984 Sep 10;259(17):10925-35
6432790
J Immunol. 2004 Aug 1;173(3):1902-7
15265923
Immunol Rev. 2009 Jul;230(1):160-71
19594635
Glycobiology. 2007 Sep;17(9):906-12
17580315
Eur J Cell Biol. 2010 Nov;89(11):788-98
20705359
Eur J Biochem. 2003 May;270(10):2109-19
12752430
Blood. 2006 Oct 1;108(7):2399-406
16778138
J Biol Chem. 1986 Aug 5;261(22):10119-26
3733704
Anal Biochem. 2004 Nov 1;334(1):36-47
15464951
Mol Biol Cell. 2010 Jan 15;21(2):219-31
19923323
J Biol Chem. 1992 Apr 5;267(10):6983-90
1313027
J Biol Chem. 2007 Jan 5;282(1):773-81
17090543
Nat Rev Immunol. 2009 May;9(5):338-52
19365409
Immunol Rev. 2009 Jul;230(1):114-27
19594632
J Biol Chem. 1989 Apr 5;264(10):5720-35
2466830
Traffic. 2007 Apr;8(4):379-88
17319896
J Biol Chem. 1994 Aug 19;269(33):20807-10
8063692
J Biol Chem. 1993 Jul 15;268(20):14940-7
8325871
Glycobiology. 2009 Jan;19(1):16-20
18849325
Glycobiology. 2006 Apr;16(4):305-17
16319083
Glycoconj J. 2004;19(7-9):433-40
14758066
J Biol Chem. 2003 Apr 4;278(14):12285-93
12538594
Biochemistry. 1991 Feb 12;30(6):1561-71
1671554
Scand J Clin Lab Invest Suppl. 1968;97:77-89
4179068
J Biol Chem. 1998 May 22;273(21):13047-52
9582341
Nat Med. 2010 Mar;16(3):295-301
20154696
J Biomol NMR. 2009 Sep;45(1-2):157-69
19641853
Biochemistry. 1999 Oct 19;38(42):13937-50
10529240
Blood. 1998 May 1;91(9):3430-8
9558402
Glycobiology. 1998 Jan;8(1):45-57
9451013
J Biol Chem. 1991 Dec 15;266(35):23648-53
1721052
Infect Immun. 2001 Feb;69(2):832-7
11159975
Biochemistry. 1993 Jan 12;32(1):260-7
8418845
Cancer Res. 1997 Dec 1;57(23):5272-6
9393748
Glycobiology. 2008 Oct;18(10):750-60
18701722
Biochem Soc Trans. 2008 Dec;36(Pt 6):1472-7
19021578
Traffic. 2009 Oct;10(10):1405-13
19650851
Cell Microbiol. 2010 Apr 1;12(4):530-44
19951367
J Biol Chem. 1993 Jul 15;268(20):14932-9
8325870
Cancer Res. 2005 Aug 15;65(16):7292-300
16103080
J Leukoc Biol. 2005 Aug;78(2):462-70
15941779
Biochemistry. 1993 Nov 30;32(47):12694-704
8251489
Expert Rev Mol Med. 2008 Jun 13;10:e17
18549522
J Biol Chem. 1986 Sep 25;261(27):12796-806
3462190
Cell Microbiol. 2002 Oct;4(10):649-62
12366402
J Mol Biol. 2005 Aug 26;351(4):695-706
16051274
J Biol Chem. 2008 Apr 11;283(15):10109-23
18216021
J Biol Chem. 1987 May 25;262(15):7383-90
2438278
J Am Chem Soc. 2005 Feb 16;127(6):1737-43
15701008
J Immunol Methods. 1999 Dec 17;232(1-2):3-14
10618505
Glycobiology. 2008 May;18(5):384-94
18263896
Nat Rev Microbiol. 2009 Jun;7(6):424-38
19444247
J Biol Chem. 2003 Feb 28;278(9):7469-75
12499376
Nat Immunol. 2007 Aug;8(8):825-34
17589510
Nat Rev Cancer. 2005 Jan;5(1):29-41
15630413
Glycoconj J. 2001 Mar;18(3):231-43
11602807
Structure. 2004 Apr;12(4):689-702
15062091
Mol Biol Evol. 2004 Jul;21(7):1177-87
14963092
J Immunol. 1999 Nov 15;163(10):5592-8
10553088
J Cell Biol. 2005 Oct 24;171(2):373-81
16247033
J Cell Sci. 2008 Feb 15;121(Pt 4):458-65
18211959
Biochim Biophys Acta. 2006 Apr;1760(4):616-35
16478649