Lactosomes: structural and compositional classification of unique nanometer-sized protein lipid particles of human milk

Abstract

Milk fat globules (MFGs) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid-protein particles termed "lactosomes" that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation, and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma high-density lipoproteins (HDL) (d > 1.063 g/mL) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk, day 8, were found to be similar in size as those from mature milk >28 days, averaging ∼ 25 nm in diameter. In total, 97 nonredundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1-laden reconstituted HDL that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition, and function between lactosomes and MFG, suggesting that these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid-protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant but to play unique, protective, and regulatory roles.

Figure 1

Images of milk fat lgobules (MFG) and lactosome fractions. (A) Scanning electron microscopy of the MFG fraction at 169 days postpartum. (B) Transmission electron microscopy of the lactosome fraction at 169 days postpartum.

Figure 2

The size distribution of lactosomes derived from early and mature milks from various donors. Each box plot represents the distribution of repeated particle diameter measurement using a commercial dynamic light scattering device. The first four digits denote the sample donor (1000, 1004, 1005) while the final two indicate the postpartum day after labor (08, 09 or 28).

Figure 3

Differences in protein abundance between milk fat globules (MFG) and lactosome fractions. The relative abundances of proteins differbetween the MFG and lactosome fractions (marginal significance, unadjusted P ≤ 0.05). Two of these proteins, immunoglobulin heavy constant alpha 1 (IGHA1) and carbonic anhydrase 6 (CA6), were significant after a multiple testing correction (B-H adjusted P ≤ 0.05).

Figure 4

ESI FT-ICR MS spectra of milk fat globule (MFG) and lactosome fractions from sample 1008 day 8 showing marked distinctions. (A) Positive mode spectrum of the MFG fraction. The MFG spectra are dominated by triglyceride (Tg, marked with a circle) and dimers of long chain fatty acids (marked with a triangle). Lipid heterogeneity can clearly be seen in the Tg species. (B) Negative mode spectrum of the lactosome fraction showing phospholipid (Pl, marked with a star) and fatty acid dimers (marked with a triangle). The phospholipids identified in this spectrum were phosphatidylinositol (m/z 890-920), bisphosphotidylinositol (m/z 670-700), and phosphatidic acid (m/z 640-650).

Figure 5

Negative mode spectrum of human milk fat globule (MFG) gangliosides for 1005d28. Legend in the figure lists the assignment of the major detected structures shown.

Figure 6

Cholera Toxin Binding Assay. (A) Positive and negative controls for the cholera toxin binding assay. Dimyristoylphosphatidylcholine (DMPC) vesicles and rHDL (containing DMPC and apolipoprotein A-I) were prepared for comparative analyses with the milk fat globule (MFG) and lactosome fractions. Positive controls were laden with 1mol% GM1 (toxin receptor) while negative controls did not contain any GM1. The graph shows the percent drop in DPH fluorescence at 490 nm for each control sample. (B) Cholera toxin binding in response to MFG and lactosomes. The percent drop in donor emission (DPH) after introduction of FITC-labeled cholera toxin is shown here. The lactosome fractions do not show a significant reduction relative to the negative controls while the MFG fractions are comparable to the positive controls.