About the Lectin

This mushroom lectin is highly specific for Galα1,3Gal
and also recognizes the porcine xenotransplantation epitope
(Galα1,3Galβ1,4GlcNAc) and the blood group B determinant ¹ .
This blood group B lectin, present in the mushroom Marasmius
oreades , is available in its native and recombinant forms ². It
is an ideal reagent for the detection and identification of the
Galα1,3Gal and Galα1,3Galβ1,4GlcNAc/Glc epitopes present
on chain ends of glycoproteins (e.g. porcine tissues and organs,
bocine thyroglobulin, 3T3cells, murine laminin, Ehrlich ascites
tumor cells) and glycolipids ³ . It is greatly more specific for
the above di- and tri-saccharides than the GS 1-B ₄
isolectin
which recognizes α-Gal end groups in any linkage. The lectin
is available as a homogeneous, water-soluble protein, in its
fluorescein- or biotinylated-forms or conjugated to agarose for
use as an affinity matrix ³ .

In examining the binding of a series of analogues of
the blood group B-trisaccharide, alphaGal(1-3)[alphaFuc(1-
2)]betaGal-OR (1, R = (CH2)8COOMe). MOA was biotinylated
and immobilized on a micro column (9.8 microL) for evaluation
by Frontal Affinity Chromatography-Mass Spectrometry
(FAC-MS) [2]. The trisaccharide 1 was found to be the epitope
needed for maximum recognition (Kd = 3.6 microM). A series
of synthetic deoxygenated and O-methylated analogues of the
B-trisaccharide (R = OMe) were then screened against the lectin,
and the key structural elements for binding were determined.
OH-4 of the beta-Gal residue and OH-2 of the alpha-Gal residue
were found to be critical for recognition. ⁴

The binding properties of MOA and GS I-B(4) to the
xenogenic disaccharide (Galalpha1-3Galbeta1) were comparable
while the binding of MOA to the xenogenic pentasaccharide
was much stronger than the binding of GS I-B(4) to the same
epitope. Non-xenogenic disaccharide-coupled neoglycoproteins
having galactose end groups linked alpha1-2 or alpha1-4 to Gal
or linked alpha1-3 to GalNAc bound very weakly to MOA,
whereas GS I-B(4) recognized all of these disaccharides with
similarly high affinity. MOA also showed high affinity for
laminin. The results indicate that the Marasmius oreades lectin
has nearly the same affinities as does GS I-B(4) for the simple
xenogenic carbohydrate antigens, but MOA has greater affinity
for the pentasaccharide and is far more specific in its binding
preferences than the Griffonia lectin. ⁵

Investigation of the structure-function relationship of
MOA defined the number and location of its carbohydratebinding
sites. Based on the sequence alignment of MOA to the
ricin B-chain lactose-binding sites, we systematically constructed
mutants by site-directed mutagenesis. Among amino acid
residues at the putative carbohydrate-binding sites, Gln(46) in
the alpha subdomain and Trp(138) in the gamma subdomain
have been identified to be important amino acid residues
directly or indirectly involved in carbohydrate recognition.
By surface plasmon resonance, Q46A and W138A were
2.4- and 4.3-fold less active than that of the wild-type MOA
(K(a) = 2 x 10(7)), respectively. A double-site mutant (Q46A/
W138A) had activity similar to W138A. The C-terminal
deletion mutant MOADeltaC showed hemagglutination
and precipitation activity, although its binding constant was
12.5-fold less active (K(a) = 1.6 x 10(6)) than that of the wildtype
MOA. A C-terminal deletion mutant with mutations
at both Gln(46) and Trp(138) (MOADeltaC-Q46A/W138A)
was 12,500-fold less active (K(a) = 1.6 x 10(3)) than that of
the wild-type MOA. Both alpha and gamma subdomains are
most probably involved in carbohydrate binding, but the beta
subdomain appears to be inactive. ⁶

REFERENCES

1. Winter, H.C. et al. (2002). J. Biochem. 277: 14996-15001.
2. Kruger, R.P., et al. (2002). J. Biochem. 277: 15002-15005.
3. Teneberg, S., et al. (2003). Glycobiology. 13: 479-486.
4. Rempel BP, Winter HC., et al. Glycoconj J. 2002 Mar;19(3):175-80.
5. Kirkeby S, et al,Xenotransplantation. 2004 May;11(3):254-61.
6. Tateno H,et al. Arch Biochem Biophys. 2004 Jul 1;427(1):101-9.