Structural Aspects of the Bovine Milk Fat Globule Membrane

Abstract

This research provides further understanding of the native structure of the bovine milk fat globule membrane (MFGM) and the role of structure on releasing from, and binding of volatile organic compounds (VOCs) to native milk fat globules (MFGs). The MFGM is constructed from a backbone of phospholipids and proteins that encapsulate the triacylglyceride core. The MFGM is believed to play an important role in many physiological processes (as a natural vehicle for delivering nutrients from the mother to the mammalian neonate). The bio-functionalities of the MFGM are regulated, not only by individual functional molecules (e.g. phospholipids and membrane proteins), but also by their specific structural organization. To date, the native structure and organization of the phospholipids and proteins are not fully understood. The research goals of this work are to extend the knowledge on MFGM protein organization (Chapter 3) and to develop an updated version of the topology of lipid organization within the MFGM (Chapter 4). Cluster of differentiation 36 (CD 36) and fatty acid-binding protein (FABP) were more strongly bound to the MFGM under the destructive forces applied to the MFGM through washing and centrifugation. The detailed localization of phospholipids and cholesterol in the MFGM is illustrated in a newly designed schematic model system in which phosphatidylcholine (PC) and sphingomyelin (SM) are located at the outer leaflet of the membrane bilayer, and phosphatidylinositol (PI) and phosphatidylserine (PS) are relatively enriched in the inner monolayer. By using confocal laser scanning microscopy (CLSM), detailed structural damage to the MFGM through common mechanical processing conditions (centrifugal washing processes) was observed. An artificial bilayer membrane model system, namely giant unilamellar vesicles (GUVs), was constructed for mimicking the biological surface morphology of the native MFGM (Chapter 5). By characterizing GUV membranes with controlled composition, evidence was provided that the lipid domains on the native MFGM were not necessarily formed from an association of sphingomyelin and cholesterol only, as previously reported in the literature, but also from individual high phase transition temperature (higher than room temperature) phospholipids, such as dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin. The GUV system is a powerful tool for studying the morphology and functional properties of biological membranes on a much larger scale than the native MFGM, allowing observation using CLSM of the phase separation of lipids on the GUV surface. The small size of native milk fat globules usually precludes this type of observation due to the poor resolution of CLSM. The GUV system is also an ideal tool for investigating the detailed functionalities of individual MFGM phospholipids in regulating the lateral segregated lipid domains on MFGs surfaces (Chapter 6). By manipulating the molar ratio between sphingomyelin and cholesterol in GUV bilayers, it was shown that cholesterol is a key constituent in regulating lipid domain formation in MFGM and that phosphatidylcholine may also interact with cholesterol in the MFGM, contributing to lipid domain formation. During the course of this research, an innovative technique was developed further to investigate interactions between VOC and native milk fat globules using confocal Raman microscopy (Chapter 7). Selected VOCs with different chemical characteristics, such as hydrophobicity, were used as probes to examine the structural feature of the MFGM and to investigate physicochemical functional properties (Chapter 8).