MODIFICATION BY SITE-DIRECTED MUTAGENESIS OF THE SPECIFICITY OF ERYTHRINA-CORALLODENDRON LECTIN FOR GALACTOSE DERIVATIVES WITH BULKY SUBSTITUENTS AT C-2
R. Arango et al., MODIFICATION BY SITE-DIRECTED MUTAGENESIS OF THE SPECIFICITY OF ERYTHRINA-CORALLODENDRON LECTIN FOR GALACTOSE DERIVATIVES WITH BULKY SUBSTITUENTS AT C-2, FEBS letters, 330(2), 1993, pp. 133-136
Examination of the three-dimensional structure of Erythrina coralloden
dron lectin (ECorL) in complex with a ligand (lactose), the first of i
ts kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862 866]
, revealed the presence of a hydrophobic cavity, surrounded by Tyr108
and Pro134-Trp135, which can accommodate bulky substituents such as ac
etamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Co
mparison of the primary sequence of ECorL with that of soybean aggluti
nin, specific for galactose and its C-2 substituted derivatives, and o
f peanut agglutinin, specific for galactose only, showed that in soybe
an agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Se
r-Trp, whereas in peanut agglutinin, the former residue is replaced by
Thr and the dipeptide by Ser-Glu-Tyr-Asn. Three mutants of ECorL were
therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser
Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the doubl
e mutant L2; Y108T. They were expressed in Escherichia coli, as done f
or recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutan
ts had the same hemagglutinating activity as native or rECorL. Their s
pecificity for galactose, GalNAc and MebetaGalNDns was examined by inh
ibition of hemagglutination and of the binding of the lectin to immobi
lized asialofetuin; in addition, their association constants with Melp
haGalNDns and MebetaGalNDns were measured by spectrofluorimetric titra
tion. The results showed that Y108T had essentially similar specificit
y as the native and recombinant lectins. The affinity of L2 and L2;Y10
8T for galactose was also the same as ECorL, but they had a lower affi
nity for GalNAc and markedly diminished affinity for the dansyl sugars
(up to 43 times, or 2 kcal, less). This appears to be largely due to
steric hindrance by the two additional amino acids present in the cavi
ty region in these mutants. Our findings also provide an explanation f
or the inability of PNA to accommodate C-2-substituted galactose deriv
atives at its primary subsite.