In Vitro Studies on Zinc Binding and Buffering by Intestinal Mucins

Abstract

The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.

A Guide to Human Zinc Absorption: General Overview and Recent Advances of In Vitro Intestinal Models

“…Subsequent animal studies confirm this hypothesis and even indicate zinc buffering properties of this physical barrier [211][212][213]. In vitro, gastrointestinal mucins bind zinc with a physiologically relevant affinity, showing a dissociation constant of the mucin/zinc-complex in the same order of magnitude as luminal zinc [103]. Since the mucus layer is not static, but represents a dynamic and viscoelastic gel [214,215], these glycoproteins might assist zinc transport to the underlying epithelium.…”

“…Detailed processes of cellular distribution of zinc into enterocytes and its transfer through the cells after its absorption are not yet completely understood. Examinations of free zinc (pools) in enterocytes in vitro with the eCalwy biosensor [101] and the fluorescent zinc probe Zinpyr-1 [102,103] document that enterocytes contain at least two different free zinc pools that are involved in the maintenance of zinc homeostasis during zinc absorption: cytoplasmic-free zinc and vesicular zinc [101][102][103]. Nevertheless, these processes have to be further scrutinized.…”

“…Three main zinc pools in enterocytes have been described: (i) cytoplasmic-free zinc, which is only complexed by low molecular weight ligands, (ii) protein-bound zinc, depicted here as metallothionein (MT)-bound zinc, and (iii) free zinc stored in vesicles [104]. The vesicular [102,103] and cytoplasmic-free zinc pools [101] are recognized to be involved in zinc absorption by enterocytes [105]. Cellular zinc homeostasis is maintained by three main groups of proteins: the zinc transporter (ZnT)-and the Zrt-, Irt-like protein (ZIP)-family as well as the zinc-binding metallothioneins [99].…”

Zinc Nutrition and Inflammation in the Aging Retina

“…Studies have showed that the microbiota have high‐affinity binding and transport systems for zinc, and there is zinc competition between microbiota in the gastrointestinal tract of a host . For example, it has been shown that excess dietary zinc (Zn) substantially alters the gut microbiota, which in the context of colonization by pathogenic bacteria, such as Clostridium difficile , may exacerbate C. difficile– associated disease by increasing toxin activity and altering the host immune response . Regarding factors that may influence zinc absorption and local zinc availability, it has been shown that the presence of intestinal mucins facilitates in vitro zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium .…”

“…For example, it has been shown that excess dietary zinc (Zn) substantially alters the gut microbiota, which in the context of colonization by pathogenic bacteria, such as Clostridium difficile , may exacerbate C. difficile– associated disease by increasing toxin activity and altering the host immune response . Regarding factors that may influence zinc absorption and local zinc availability, it has been shown that the presence of intestinal mucins facilitates in vitro zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium . In a clinical study comparing individuals with rheumatoid arthritis (RA) with healthy subjects, the redox environment, transport, and metabolism of iron, sulfur, zinc, and arginine were found altered in the microbiota of individuals with RA, along with detectable specific alterations in the gut and oral microbiome, including molecular mimicry of human antigens related to RA, in individuals with the disease .…”