C-Terminal Fragment of Vitellogenin II, a Potential Yolkin Polypeptide Complex Precursor Protein—Heterologous Expression, Purification, and Immunoregulatory Activity

Abstract

The aim of this research was to analyze the heterologous expression, purification, and immunoregulatory activity of recombinant YGP40 (rYGP40), the potential precursor of the yolkin peptide complex. The ygp40 coding sequence was codon optimized, successfully expressed in the E. coli system, and purified from inclusion bodies with a yield of about 1.1 mg/L of culture. This study showed that the protein exhibits immunomodulatory activity, expressed by the stimulation of TNF-α and IL-10 production and nitric oxide induction at a level comparable to that of the natural yolkin peptide complex obtained by other authors from hen egg yolk. At the highest dose of 100 µg/mL, rYGP40 also caused the up-regulation of iNOS expression in murine bone marrow-derived macrophages (BMDM). Moreover, no cytotoxic effects of rYGP40 on the BMDM cell line were observed.

Yolkin, a Polypeptide Complex from Egg Yolk, Affects Cytokine Levels and Leukocyte Populations in Broiler Chicken Blood and Lymphoid Organs after In Ovo Administration

“…In such conditions, yolkin enhanced the production of TNF-α, IL-1β, IL-6, and IL-10 in human whole blood cell cultures [24,25], as well as nitric oxide (NO) release by the murine macrophage cell line J774.2 [25] and murine-bone-marrow-derived macrophages (BMDM) [27]. Moreover, similar effects were provided by a potential precursor of yolkin, recombinant YGP40, which increased TNF-α, IL-10, and NO synthesis in human whole blood cells and murine-bone-marrow-derived macrophages [35]. On the other hand, Obmi ńska-Mrukowicz et al [29] reported that yolkin administered to mice diminished ex vivo NO synthesis by peritoneal macrophages stimulated with lipopolysaccharide (LPS).…”

“…Regarding cytokines, there are literature reports on the stimulatory effects of yolkin on the secretory activity of cells from monocyte-macrophage cell lineage in vitro [24,25,[27][28][29]35]. In such conditions, yolkin enhanced the production of TNF-α, IL-1β, IL-6, and IL-10 in human whole blood cell cultures [24,25], as well as nitric oxide (NO) release by the murine macrophage cell line J774.2 [25] and murine-bone-marrow-derived macrophages (BMDM) [27].…”

Importance for humans of recently discovered protein compounds – yolkin and yolk glycopeptide 40, present in the plasma of hen egg yolk

“…The polypeptides of about 4 and 12 kDa are free of carbohydrates and start at position 1732 in the Vt amino acid sequence, whereas the other larger protein fractions (16, 19, 23, 29, 32, and 35 kDa) are glycoproteins corresponding to the amino acid sequence of Vt starting at position 1572 ( Polanowski et al, 2013 ) ( Figure 1 ). On this basis, it can be assumed that Y may be considered as a set of peptides generated during proteolytic cleavage of YGP40 at positions 1571S-1572A and 1731R-1732M ( Szmyt et al, 2021 ). Based on this, it can be supposed that the protein profile of Y is most likely influenced by the activity of cathepsin D degrading Vt during egg formation.…”

“…Limited information is available about the biological properties of YGP 40. Recently, Szmyt et al (2021) produced YGP40 using a recombinant DNA technology involving genetically modified E. coli BL21 (DE3) expression cells. It has been shown that recombinant YGP40 possesses immunomodulatory activity and can stimulate human whole blood cells to produce TNF-α and IL-10 and induce nitric oxide production.…”

“…Pro-inflammatory TNF-alpha, secreted by Th1 cells, controls cellular immunity, while anti-inflammatory cytokine IL-10 secreted by Th2 cells is responsible for the control of the humoral immune response. rYGP40 also causes the up-regulation of iNOS expression in murine bone marrow-derived macrophages ( BMDM ) and has no cytotoxic effects on BMDM ( Szmyt et al, 2021 ).…”