Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion

Abstract

Key points: Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules.

Abstract: Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion.

Keywords: LC_MS/MS; Xanthine Oxidoreductase; apocrine secretion; lactation; milk fat globule; proteomics.

Figure 1. Conditional knockout models of XOR in the mammary gland show a modest lactation defect

A, H&E‐stained FFPE tissue from dams at lactation day 10–12 with the following genotypes: XOR^flox/flox, XOR^flox/flox, MMTV‐Cre^+/−, XOR^flox/flox, WAP‐Cre^+/− and XOR^flox/flox, BLG‐Cre^+/−. Luminal spaces are marked with an L. Arrows indicate small lipid droplets docked at the apical plasma membrane (APM) of the mammary epithelial cell (MEC). Asterisks indicate large lipid droplets within the MEC in the XOR MGKO glands. Bar = 100 μm. B, litter growth curves for lactation days 0–2 shown for XOR^flox/flox (WT) dams, XOR^flox/+, BLG‐Cre^+/− (HEMI) dams and XOR^flox/flox, BLG‐Cre^+/− (MGKO) dams. C, litter weights for eight pups, the morning after birth. (^a,b P<0.05). D, litter growth curves for lactation days 0–12 for the same groups. E, litter growth rates for days 3–12 for the above groups (^a,b P<0.005). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. XOR mediates close association between the cytoplasmic lipid droplet and the apical plasma membrane

Electron micrographs of lactating mammary gland from WT (A–D) and MGKO (E–H) dams. A and D, a cytoplasmic lipid droplet (CLD) near the apical plasma membrane (APM). Bar = 1 μm. B and F, enlargement of inset delineated with dotted line. C and G, alveolar cell with several CLDs near at APM. Bar = 5 μm. D and H, secreted MFGs in lumen with attached crescent. Bar = 5 μm. The luminal space is marked with an L. Arrows delineate distance from CLD to plasma membrane. White arrowheads indicate secretory vesicles containing casein micelles. Secreted MFGs are labelled with an asterisk (^*). Those with crescents are marked with double asterisks (^**).

Figure 3. XOR draws Btn into the membrane domain where CLDs are docked

A, L10 mammary gland from WT and XOR MGKO dams immunostained for Btn (right, and red), Plin2 (middle, and green) and overlay with DAPI (blue). Arrows indicate docked CLDs in the WT gland. Asterisks indicate large CLDs within MGKO glands. Bar = 10 μm. B, line intensity analysis of CLD docking showing peak‐to‐peak distance between Btn and Plin2 at the membrane (>50 CLD per mouse, three mice per group, P<0.0001). C, L10 mammary gland from WT and XOR MGKO dams stained with anti‐Btn (red), WGA (green) and DAPI (blue), showing Btn (red) staining is clustered in the WT membrane (WGA, green) but not in the MGKO membrane. Bar = 10 μm. Dotted line indicates enlarged region shown as a 3D projection. Movies show 3D imaging of CLD docking sites in WT and MKGO glands. D, line intensity analysis of CLD docking showing segregation of Btn from WGA staining. Pearson's correlation coefficient shows anti‐correlation of Btn and WGA in the docked CLD in WT glands (−0.35 ± 0.4) but not in the XOR MGKO glands (0.13 ± 0.13, P = 0.0004). Fifty CLDs per group were scored from three animals per group.

Figure 4. Docking of CLDs to the membrane disassembles the microvilli and remodels the glycocalyx

A, 3D projections of CLDs at the apical plasma membrane (APM) of WT and MGKO glands, stained with anti‐Plin2 (green), WGA (red) and DAPI (blue). Arrows indicate the angle of membrane inclusion. B, diagram of angle of inclusion measurements performed on docked CLDs. C, angle of inclusion measured in CLDs docked at the APM of WT and MGKO mammary glands (combined data from three mice per group, >50 CLDs per mouse). D, electron micrograph of a docked CLD in a WT gland with the membrane colourized (red) to show microvilli and membrane stretch. The luminal space is marked ‘L’ and a secreted MFG in the lumen is marked with an asterisk. E, line intensity analysis of CLDs on Plin2/WGA‐stained mammary glands. Peak‐to‐peak distance is increased with XOR loss. WGA staining of the membrane over the docked CLD is decreased in the WT but not the MGKO gland (>50 CLDs per mouse, three mice per group, P<0.0001).

Figure 5. CideA mediates CLD fusion and docking

A, L10 mammary gland from WT and XOR MGKO dams immunostained for CideA (red, right), Plin2 (green, centre) and DAPI (blue in overlay). Arrowheads delineate docked CLDs with CideA clustered. Asterisk marks a large CLD that is not docked. Bar = 10 μm. B, 3D projection view of fusing CLDs. CideA (right, red), Plin2 (centre, green) and overlay showing CideA concentrated at the fusion pore of two CLDs in WT and MGKO glands. Bar = 2 μm. C, pre‐secretion CLD sizes measured in the cytoplasm near the apical plasma membrane (APM) in Plin2‐stained WT and XOR MGKO cells (>50 CLDs per mouse, three mice per group).

Figure 6. Very large lipid droplets are secreted into the milk of XOR MGKO glands

A, crematocrit (volume % milk fat) of milk collected from WT (36 ± 9) and MGKO (50 ± 7) dams at lactation day 10 (mean ± SD, P = 0.0003). B, BODIPY 493/503‐stained, post‐secretion MFGs (left). Arrows indicate MFGs damaged in the milking process. LipidTox Red and Alexa 488‐phalloidin (green) staining of globules from WT and XOR MGKO milk (right). Bar = 10 μm. C, diameter of post‐secretion MFGs, stained with BODIPY 493/503 (>800 MFGs per mouse, three mice per group). Size ranges are displayed as a fraction of the total counted and as a fraction of the total lipid volume.

Figure 7. Volcano plot reveals asymmetry in proteins associated with MFGs from XOR MGKO dam

Plot of the log₂fold change (WT/MGKO) of the spectral abundance versus the log₁₀(P‐value) for the proteome of the MFGs. The empty point is XDH/XOR (marked with an x). The most significantly changed proteins, all in greater abundance on MGKO MFGs are: a, glutamyl‐prolyl‐tRNA synthetase (EPRS), 26‐fold increased; b, lipopolysaccharide (LPS)‐binding protein (LBP), 6.2‐fold increased; c, Pnpla2, a.k.a. adipose triglyceride lipase (Atgl), 4.7‐fold increased; d, tubulin alpha 1b (Tuba1b), 11‐fold increased. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 8. String networks of proteins significantly changed on MFGs from WT and XOR MGKO dams reveal interacting proteins

A, only eight proteins are significantly lost from the MFGs when XOR (XDH) is genetically deleted (see Table 2). XOR is known to interact with Btn. Fgg, Fgb and Apoc3 also interact. B, 52 proteins are significantly increased on the MFGs when XOR is deleted. Labelled are nodes related to vesicle trafficking, lipid metabolism and cytoskeleton. [Colour figure can be viewed at wileyonlinelibrary.com]