An Accumulation of Caseins in the Endoplasmic Reticulum Due to as-1 Casein Deficiency Induces a Chronic ER Stress

Beauvallet C., Bevilacqua C., Badaoui B., Cebo C., Makhzami S., Chanat E. and Martin P.
Génomique & Physiologie de la Lactation, INRA Jouy en Josas, France

The extensive polymorphism recorded at the CSN1S1 locus influences the composition of goat milk and its technological properties (milk clotting ability, organoleptic properties, …).

The absence (or reduced amounts) of αs1-casein is responsible for the accumulation of immature caseins in distended rough endoplasmic reticulum cisternae and consequently for a perturbation of the whole secretion process (protein and lipids). In contrast, no accumulation was found in mammary epithelial cells lacking b-casein expression (CSN2 O/O). This suggests that as1-casein interacts with the other caseins and that this complex is required for their transport to the Golgi. To understand the underlying mechanisms and go further in the characterization of cell functioning (and dysfunction) we have first performed comparative gene expression profiling experiments (transcripts and proteins), starting from mammary tissues and epithelial cells isolated from goats of extreme genotypes (CSN1S1 O/O versus CSN1S1 A/A).

Microdissected acini from both genotypes were drawn and directly sticked on a Maldi-MS plate. A minute volume of matrix solution was deposited onto the cells and the plate was inserted into the Maldi-Tof spectrometer. Several spectra were acquired in different ranges, recorded and compared. Some peaks were found to give different intensity levels, specifically in the casein region.

Microsomal fractions were prepared from lactating goat mammary glands of both genotypes to reduce sample complexity and yield ER protein profiles using high-resolution 2D-gel electrophoresis/mass spectrometry analysis. Given the increased expression of BiP in the ER of mammary tissue from goats CSN1S1 O/O, experiments targeting genes involved in the unfolded protein response (UPR) were designed to test the hypothesis of an ER stress induced by the accumulation of immature caseins.

In addition, a differential proteomic analysis of milks from goats of the same genotypes has been undertaken. The Differential Gel Electrophoresis (DIGE) technique was performed on 2 sets of 4 samples, one of milks from wild-type goat, the other from as1-casein deficient goat milks. Micellar caseins were removed by centrifugation and the supernatants labelled with the three Cy dyes, of which one (Cy2) was used as internal standard (an equimolar mix of all the samples). Two-dimensional electrophoresis gels were scanned with a three-laser imaging system, delivering 3 images each, which were analyzed with the software SameSpots. Statistically, about 95 spots were significantly discriminated and the most powered spots were identified by Maldi-Tof-MS. The occurrence of ER-resident proteins in O/O milks strongly suggests a different secretory mechanism for this genotype.