C. roseum lectin (CRL) was purified by a single step using a Sepharose-4B-mannose affinity chromatography column (Fig. 2
a). All purification steps were monitored by haemagglutinating activity and SDS–PAGE.
Figure 2 Affinity chromatography and SDS–PAGE of the purified C. roseum lectin (CRL). (a) Sepharose-4B-mannose chromatography. (b) SDS–PAGE showing protein markers (left) and a purified CRL band (right). The molecular-weight markers are bovine (more ...)
CRL showed haemagglutinating activity towards papain-treated, trypsin-treated and untreated rabbit erythrocytes. The minimal concentration of purified protein that agglutinated a 2% rabbit erythrocyte suspension was <2 µg ml−1
. This haemagglutinating property is similar to those of other Diocleinae lectins. From the inhibitory substances tested, mannose was the most potent, with a minimum concentration of 19.5 mM
. The CRL N-terminal sequence was found to be ADTIVAVELDSYPNTDIGDPSYPH. This sequence is very similar to those of other lectins (Table 1) from the subtribe Diocleinae [100% identity with Dioclea lehmanni
I (Perez et al.
) and Canavalia brasiliensis
(Moreira & Cavada, 1984
); 92% identity with D. grandiflora
(Moreira et al.
), ConA (Hague, 1975
) and Cratylia floribunda
(Oliveira et al.
)]. The apparent molecular weight of CRL was determined by SDS–PAGE after heating in the presence of SDS (Fig. 2
). The lectin appears to be composed of three polypeptide chains of approximate molecular weights of 30, 18 and 12 kDa. This variety of molecular weights is similar to that found for other lectins from Diocleineae species such as Canavalia ensiformis
). This latter lectin, commonly known as concanavalin A, has been shown to initially be expressed with an initial set of termini, which led to the supposition that the gene product is then cleaved post-translationally to form two chains and a second set of termini and then fused into a single chain again by peptide-bond formation that joins the initial set of termini, a kind of circular permutation of the sequence. In the absence of other evidence, we suppose that the smaller chains are analogous to the two chains of the cleaved gene product and the largest chain is analogous to the fused final product (Cunningham et al.
N-terminal sequence alignment of Diocleinae lectins
Microcrystals were obtained using 0.1 M
HEPES pH 7.5 containing 10%(w
) PEG 3350 and 0.2 M
proline. Improvement of this crystallization condition was obtaining by increasing the pH and PEG concentration. Suitable crystals were obtained from drops containing 0.1 M
Tris–HCl pH 7.8, 8%(w
) PEG 3350 and 0.2 M
proline. CRL crystals grew within a month to maximum dimensions of approximately 0.8 × 0.4 × 0.4 mm (Fig. 1
). The diffraction data showed the CRL crystals to be orthorhombic, belonging to space group P
, with unit-cell parameters a
= 67.8, b
= 103.1, c
= 122.1 Å. CRL crystal data were scaled in the resolution range 34.92–1.77 Å. Statistics of the data collection can be found in Table 2. Assuming the presence of four molecules of 25 kDa (as is standard for Diocleinae lectins that present an apparent weight of 30 kDa in SDS–PAGE, such as ConA and D. lehmanni
lectin I) in each monomer in the asymmetric unit, the calculated V
was 2.1 Å3
), indicating a solvent content of 41.9%. Elucidation of the complete amino-acid sequence of CRL, now in progress, will permit the refinement of a three-dimensional CRL structure.
Summary of data-collection statistics for CRL