A Native-MS Study of Charge-Driven Interactions of Glycosaminoglycans with Lactoferrin Underlying Its Immunoprotective and Immunomodulatory Properties

Apart from its role as a nutrient in mammalian milk, lactoferrin (LF) is also an important part of the innate immune system, where it functions as a potent microbicidal molecular factor. An important structural feature of LF, which makes it distinct from its next-of-kin serum transferrin (TF, an iron transporter that is not involved in the innate immune response), is the presence of an extended patch of positive charge on the surface of LF. While the relevance of this structural feature to the protein's immunoprotective properties is indisputable, the specific molecular mechanism that governs its involvement in protection against a wide array of pathogens remains poorly understood. We use native mass spectrometry (MS) and molecular modeling to study LF interaction with glycosaminoglycans, whose structure approximates that of the highly sulfated segments of heparan sulfate (HS), a major component of the extracellular matrix. Even the shortest highly sulfated HS segments readily associate with LF under near-native conditions, suggesting that this interaction plays an important role in concentrating the microbicidal agent in the vicinity of the point of its release from the activated neutrophil, thereby preventing its removal from the infection site by diffusion or blood flow. No such properties are exhibited by TF, consistent with its role as an iron transporter that needs to be freely circulated until its encounter with a receptor on the surface of a cell that requires a supply of iron for its growth or specific function(s). We also examine the interaction of LF with heparin, a highly sulfated glycosaminoglycan that is released from the mast cells upon their activation. Native MS reveals the ability of a single heparin chain to accumulate a significant number of LF molecules (up to five), consistent with its proposed role as an antagonist of the heparin-associated tryptases (which are deactivated upon their dissociation from heparin). This provides a molecular basis for the immunomodulatory properties of LF as a factor limiting the harm to the host inflicted by tryptases in the course of the mast cells' coactivation with neutrophils.