Pseudomonas aeruginosa Lectin PA-IIL as a Powerful Probe for Human and Bovine Milk Analysis

Abstract

Milk composition exhibits species-specific differences depending on genetic, evolutionary, and environmental factors. In addition, commercial milk preparations are also changed by industrial manipulations, including severe heat processing. Cow milk, used as human food, provides important nutrients but lacks some essential components that are present in raw human milk. The present study, which was aimed at comparing infant breastfeeding to cow-based formula nourishment, shows major differences between the human and the commercial cow milk glycans detectable by the lectins PA-IL (galactose-binding) and PA-IIL (fucose and mannose-binding) isolated from the cells of human pathogen Pseudomonas aeruginosa. More than 40 human milk samples, several cow milks, and bovine milk-based infant formulas, were examined using these two lectins. For purposes of comparison, the plant lectins Concanavalin A (Con A), which binds mannose, and Ulex europaeus 1st lectin (UEA-I), which binds fucose, were also used. The most prominent difference was revealed using PA-IIL, which displayed a unique high sensitivity to the human milk fucosylated compounds. PA-IL and UEA-I also exhibited preferential sensitivity to the human milk but considerably lower than that of PA-IIL. Con A was inhibited by human and the other milk preparations examined to the same extent. These findings indicate the superb applicability of PA-IIL for rapid and reliable comparative investigation of milk glycans from human and cow, indicating which glycans could be added to infant formulas in order to enrich them, as well as for verification and quality control of otherwise improved bovine milk-based infant formulas.

Analyses of diverse mammals’ milk and lactoferrin glycans using five pathogenic bacterial lectins

“…The latter aspect was studied due to our previous findings of profound differences between human and cow milk abilities to block PA-IIL and CV-IIL. Human milk exerted much stronger inhibition and its Wb analyses revealed very heavy lactoferrin band staining by these two lectins (Lesman-Movshovich & Gilboa-Garber, 2003;Zinger-Yosovich et al, 2010). The special interest in lactoferrin was also associated with its being a multifunctional (iron-binding) Gp displaying potent anti-bacterial, -fungal, -protozoal and -viral activities (Lonnerdal, 2009;Valenti & Antonini, 2005), also including hampering dendritic cell (DC)-mediated HIV-1 transmission to T-cells by blocking the binding of gp120 to DC-SIGN Naarding et al, 2005).…”

“…In contrast to human milk, the animal ones contain both N-acetyl (Neu5Ac)-and N-glycolyl-neuraminic acid but their repertoire of Fuca1-2/3/4-bearing oligosaccharides and GPs is relatively poor. These differences may be detected and differentiated by means of lectins (Lesman-Movshovich & Gilboa-Garber, 2003) from plants, animals and microorganisms, which are widely used for investigations and comparisons of different natural glycans, including GPs, for diverse scientific and applicative (biotechnological, agricultural and medical) purposes. Lectins isolated from pathogenic bacteria have been shown to be as useful as the other ones for those applications.…”

“…For example, purified Pseudomonas aeruginosa (P. aeruginosa) fucose-binding lectin PA-IIL (Gilboa-Garber, 1982) which is involved in this pathogen adhesion (Chemani et al, 2009;GilboaGarber, Avichezer, & Garber, 1997;Mitchell et al, 2002) clearly differentiates between human and bovine milks -interacting with human milk much more strongly than with cow milk. The interactions are detectable by analyses of the lectin haemagglutinating activity inhibition (HAI) and Western blotting (Wb) of the milks using peroxidase-labelled lectins (Lesman-Movshovich & Gilboa-Garber, 2003). Gustafsson, Kacskovics, Breimer, Hammarstrom, and Holgersson (2005) used antibodies and plant lectins for Wb analyses of milks from diverse mammals including human and cow milks.…”

Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specifities

“…Additional examples include analyses of avian egg white and human saliva gps [8][9][10] by diverse lectins for studies of their glycosylations and variations in disease, and of human milk gps [1,2,11] for antimicrobial adhesion potential [2]. Usage of Pseudomonas aeruginosa fucose (Fuc)-binding lectin (PA-IIL) [12] for detection of fucosylated gps in human milks, indicated its efficiency as a powerful tool for differentiation between human and cow's milk [13].…”

“…After 30 min interaction, 50 mL of the compatible erythrocyte suspension was added to each tube and the hemagglutination was examined as above. The hemagglutination inhibition intensity was represented by the number of tubes without hemagglutination preceding its reappearance [13]. Lectin usage for investigation of human body fluids may serve diverse, important scientific, and applicative purposes.…”