The effect of carbohydrate moiety structure on the immunoregulatory activity of lactoferrin in vitro

Abstract

Abbreviations used: AFC -antibody-forming cells; bLF -bovine milk lactoferrin; ConAconcanavalin A; DegbLF -deglycosylated bovine lactoferrin; FCS -fetal calf serum; hLFhuman lactoferrin; LF -lactoferrin; LPS -lipopolysaccharide; MLR -mixed lymphocyte reaction; MTX -methotrexate; PBMC -peripheral blood mononuclear cell; rhLFrecombinant human lactoferrin; rhLF A -non-sialylated recombinant human lactoferrin; rhLF B -sialylated recombinant human lactoferrin; SRBC -sheep red blood cells University of Texas, Health Science Center, Houston, Texas, USA Abstract: The aim of this study was to evaluate the immunoregulatory effects of recombinant human lactoferrin (rhLF) in two in vitro models: (1) the secondary humoral immune response to sheep erythrocytes (SRBC); and (2) the mixed lymphocyte reaction (MLR). We compared the non-sialylated glycoform of rhLF as expressed by glycoengineered Pichia pastoris with one that was further chemically sialylated. In an earlier study, we showed that sialylated rhLF could reverse methotrexate-induced suppression of the secondary immune response of mouse splenocytes to SRBC, and that the phenomenon is dependent on the interaction of lactoferrin (LF) with sialoadhesin (CD169). We found that the immunorestorative activity of sialylated rhLF is also dependent on its interaction with the CD22 antigen, a member of the immunoglobulin superfamily that is expressed by B lymphocytes. We also demonstrated that only sialylated rhLF was able to inhibit the MLR reaction. MLR was inhibited by bovine lactoferrin (bLF), a glycoform that has a more complex glycan structure. Desialylated bLF and lactoferricin, a bLF-derived peptide devoid of carbohydrates, did not express such inhibitory activity. We showed that the interaction of LF with sialic acid receptors is essential for at least some of the immunoregulatory activity of this glycoprotein.

Overview of Lactoferrin as a Natural Immune Modulator

“…8 Higher-affinity binding of lactoferrin (Kd = 20-200 nM) to a few dozen protein cell receptors was reported. 2 Some of these receptors are lectins recognizing the glycan moiety of lactoferrin (bovine and/or human), such as the scavenger receptor C-type lectin on endothelial cells, 22 dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin on antigen-presenting cells (APCs), 23 Hepatic lectin 1 on hepatocytes, 24 Siglec-1 (CD169/sialoadhesin), 25 and TLR-4. 15 Others are cell receptors that recognize, for the most part, the Lfc or N1 lactoferrin domain.…”

Lactoferrin, a Pleiotropic Protein in Health and Disease

“…Humans Lf (hLf) may have multiple post-translational modifications such as phosphorylation (169) and enrichment with N-glycans (11) ( Table 1). Although the exact function of these modifications is not fully understood, they may protect Lf from proteolysis and are also involved in receptor recognition (11,237).…”

Immunomodulatory properties of human recombinant lactoferrin in mice: Implications for therapeutic use in humans

“…In fact, sugar moieties in LF play a crucial role in the interaction with the host's cell receptors. 22,23 Therefore, there is an urgent need for LF that is fully compatible with humans in order to avoid species-specific incompatibilities and to ensure adequate therapeutic effect.…”