Seeds contain high levels of polysaccharides (PSs), which function as substrate reserves for germination and as osmoprotectants [18
]. In addition, they may also contribute to the protection of embryos against infections by blocking pathogenic bacterial lectins [17
]. The differential interactions of the five seed extracts with the patholectins PA-IL, PA-IIL, and CV-IIL, are compiled in Figure and Tables and . The herein-examined seeds of cashew, cocoa, coffee, pumpkin, and tomato were chosen due to their edibility and their rich nonstarch PS reserves, which were thoroughly studied by experts in the field [18
]. In general, most seed PSs contain galactans, galactomannans, mannans, and xyloglucans (Table ). Among the examined seeds, those of cocoa beans were reported to be outstanding in their major, highly branched, pectic PSs (60% of the total cell-wall PSs), constructed of rhamnogalacturonan backbone heavily substituted by 5-linked Ara and 4-linked Gal side chains [19
]. In addition, they were found to contain fucosylated xyloglucan (Figure ) and galactoglucomannan [19
] consisting of 1-4-linked Glc and Man backbone, with 42% of the Man residues and 13% of the Glc residues substituted at O-6 by Gal or by several Gal pairs, with either Gal, Ara, or xylose as the terminal saccharide [19
]. The coffee-bean cell-wall PSs, which constitute half the bean dry weight [20
], contain galactomannans composed of 1-4-linked β Man backbone substituted at O-6 by single Gal residues (Table ) and type-II arabinogalactans (consisting of a 1-3-linked β-Gal backbone substituted mainly at O-6 by side chains of Gal, Ara, and Rha residues and 2 mol % of glucuronic acid residues with the Ara as the terminal residue). The latter are usually covalently linked to proteins containing 10% of 4-hydroxyproline residues [20
]. The pumpkin seeds were found to contain high mannose-type free N-glycans [22
] (Table ) and the tomato seeds contain approximately 60% Man, largely as β1-4-mannan backbone, with lesser amounts of glucose, Gal, and Ara, probably in the form of α-Gal side chain-bearing galactomannans or galactoglucomannans [23
], which decompose during germination [24
]. The blocking of the P. aeruginosa
and C. violaceum
lectins by the examined seed extracts (Figure and Table ) was compatible with the above-described composition of their glycans. The staining of the seed-extract Wbs by the peroxidase-labeled bacterial lectins has added important novel information as to their epitope-bearing GPs (Figure and Table ).
Patholectin-inhibiting glycodecoy activities in non-dialyzed (N) and dialyzed (D) seed extract preparations
Comparison of the edible seed glycodecoy inhibitory activities to those of animal embryo-protecting and neonate-protecting substances
The active seed glycodecoy epitopes involved in the blocking of PA-IL, PA-IIL, and CV-IIL (graded on an intensity scale of ± - ++++)
The possible structures of the active lectin-binding epitopes of the examined seed galactosylated (PA-IL-binding), mannosylated, and fucosylated (PA-IIL-binding and CV-IIL-binding) oligosaccharides. Binding sugars are marked in bold
Figure and Table show that P. aeruginosa
Gal-binding PA-IL, which was most sensitive to blocking by the locust- and guar-bean galactomannans [17
], was also inhibited by the cashew-, cocoa-, coffee-, pumpkin-, and tomato-seed extracts, as expected based on the documented presence of Gal-bearing glycans in all of them (Figure and Table ). The significant inhibition of PA-IL by the cocoa-seed extract is compatible with the description of the special Gal-bearing, highly branched pectic PSs and galactoglucomannans in the seeds [19
]. The highest inhibitions of PA-IL by the coffee- and tomato-seed extracts are also in line with the reports on the coffee Gal-bearing galactomannans and arabinogalactans [20
] and the tomato galactomannans and galactoglucomannans [23
Comparison of nondialyzed (containing both low MW [LMW, < 10 kDa] and high MW [HMW] glycans) to dialyzed seed extracts (retaining only the HMW [> 10 kDa] glycans) revealed that PA-IL blocking was due to both LMW and HMW saccharides (Figure ). The PA-IL-stained Wbs of the tomato seed extracts revealed one pale GP band (at around 55-60 kDa). In the cashew Wb there were 2 strong GP bands (at around 27 and 31 kDa) and a few weaker ones (at 33, 37, and 45 kDa). In the pumpkin, there were more than 10 bands between 28 and 160 kDa, with those around 30, 31, and 80 kDa being the boldest.
Interestingly, most of the PA-IL-stained cashew and pumpkin GP bands and the one tomato band were also stained by PA-IIL and CV-IIL, revealing that their GP oligosaccharides were of hybrid type, with both Gal- and Fuc- or Man-type bearing antennae.
Figure shows that P. aeruginosa
fucophilic (Fuc-, Man-, and D-Ara-binding) lectin PA-IIL was inhibited by the same five seed extracts. Its inhibition was not due to the PA-IL-blocking galactomannans (Tables and Figure ) [17
], but might be due either to terminal Fucα1-2-linked residues carried on β-Gal side chains of some xyloglucans and arabinogalactans [19
] or to high-mannose-type N-glycans in either free form (e.g., in the pumpkin seeds) [22
] or linked to macromolecules. The PA-IIL-binding GP bands might contain either oligo or high-mannose-type N-glycans or Lea
-epitope (Figure ), which is the best PA-IIL ligand [8
] that also contributes to its highest blocking by human milk (Figures , and Tables and ) [14
- bearing antennae are found in plants as short N-glycans and in association with PSs and GPs [26
]. PA-IIL staining of the examined-seed Wbs exhibited considerably more GP bands than PA-IL: at least 10 each in the coffee, pumpkin, and tomato lanes. The PA-IIL-stained pumpkin-seed Wb displayed around 15 GP bands, most of them also stained by PA-IL. However, the relative intensities of the staining of these bands by the 2 lectins were not similar: while PA-IL most strongly stained the bands at around 25-30 kDa, PA-IIL most strongly stained a band at around 33 kDa.
As seen in Figure , in contrast to the two P. aeruginosa lectins, the C. violaceum fucophilic lectin CV-IIL displayed low sensitivity to the coffee-, tomato- and pumpkin-seed glycans and was negligibly inhibited by the cashew- and cocoa-seed extracts. Three GP bands were observed in the CV-IIL-stained cashew Wb. They also interacted with PA-IL and PA-IIL. The CV-IIL staining of the 50-kDa-cashew GP was darker than that observed with PA-IIL (this band was not stained by PA-IL). The CV-IIL-stained coffee and pumpkin-seed Wbs showed weaker interactions, while in the tomato-seed Wb there were four bold bands (at around 10, 30, 32, and 60 kDa) and 2 weaker ones (at 35 and 40 kDa), all of them also seen in the respective PA-IIL-stained Wbs. Weak CV-IIL inhibitions by the seed extracts (as opposed to PA-IIL and its own blocking by the animal products [Figure and Table ]), can be ascribed to the strict selectivity of this lectin (also exhibited in its insensitivity to inhibition by the PA-IIL-blocking yeast mannan) (Tables and ). The GP bands observed in the CV-IIL-stained cashew, pumpkin, and especially tomato Wbs were not associated with considerable lectin blocking, probably due to either the low level of the GPs or low affinity to them. Lack of correlation between lectin-binding intensity and Wb-band staining is not surprising since there is generally no quantitative correlation between the intensities of these two parameters.
The shared PA-IIL and CV-IIL bands in the cashew-, pumpkin-, and tomato-seed Wbs might represent the Fucα1-2 residues linked to GPs through asparagine-bound N-glycans, as described by Puhlmann et al. [29
]. The exclusive PA-IIL-stained coffee and pumpkin GP bands, not stained by CV-IIL, confirm the higher selectivity of the latter (Table ).