Protein profiles of enzymatically isolated rumen epithelium in sheep fed a fibrous diet

Abstract

BackgroundThe rumen wall plays a major role in efficient transfer of digested nutrients in the rumen to peripheral tissues through the portal venous system. Some of these substrates are metabolised in the epithelium during this process. To identify the specific proteins involved in these processes, we used proteomic technologies. Protein extracts were prepared from ventral rumen tissue of six sheep fed a fibrous diet at 1.5× maintenance energy requirements. Using a newly developed method, we were able to enzymatically isolate the epithelial cells from underlying tissue layers, thus allowing cytosol and membrane fractions to be independently analysed using liquid chromatography tandem mass spectrometry (LC MS/MS).ResultsUsing our procedure we identified 570 epithelial proteins in the Ovis aries sequence database. Subcellular locations were largely cytosolic (n = 221) and extracellular (n = 85). However, a quarter of the proteins identified were assigned to the plasma membrane or organelle membranes, some of which transport nutrients and metabolites. Of these 91 were transmembrane proteins (TMHMM), 27 had an N-terminal signal peptide (signalP) and TMHMM motif, 13 had a glycosylphosphatidylinositol (GPI) anchor and signalP sequence, 67 had beta (β) strands or 17 β strands and a transit peptide sequence, indicating the identified proteins were integral or peripheral membrane proteins. Subunits of the 5 protein complexes involved in mitochondrial cellular energy production were well represented. Structural proteins (15%), proteins involved in the metabolism of lipids and proteins (26%) and those with steroid or cytokine action were a feature of the proteome.ConclusionOur research has developed a procedure to isolate rumen epithelium proteins from the underlying tissue layers so that they may be profiled using proteomic technologies. The approach improves the number of proteins that can be profiled that are specific to the epithelium of the rumen wall. It provides new insights into the proteins of structural and nutritional importance in the rumen epithelium, that carry out nutrient transport and metabolism, cell growth and signalling.Electronic supplementary materialThe online version of this article (10.1186/s40104-019-0314-0) contains supplementary material, which is available to authorized users.

Cross-tissue single-cell transcriptomic landscape reveals the key cell subtypes and their potential roles in the nutrient absorption and metabolism in dairy cattle

“…These three tissues have anatomically similarly stratified squamous epithelium [18] consisting of keratinocytes that include basal cells (BC), spinous cells (SC), and granule cells (GC). Multiple cell subtypes among BC (highly expressing KRT14 ) [19] , [20] , SC (highly expressing KRT10 , KRT6A , and S100A8 ) [20] , [21] , and GC (highly expressing DLK2 ) [22] were identified in each tissue. We noted that the cluster 6 spinous cell subtype in the reticulum and cluster 15 spinous cell subtype in the omasum shared common gene expression signatures with cluster 10, cluster 14, and cluster 19 spinous cell subtypes of the rumen, which specifically highly expressed GJA1 ( Fig.…”

“…Cell type definition is another major challenge in scRNA-seq studies of bovine metabolic tissues. We carefully studied and referenced previous studies [19] , [20] , [21] , [22] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] , [61] , [62] , [63] , [64] , [65] , [66] , [67] to generate a list of canonical cell-type-specific markers for each cell type with manual curation (Table S1). Moreover, due to the limited availability of markers with specificity to bovine cell lineages, many of the markers in the list of canonical cell-type-specific markers were drawn from murine and human datasets.…”

Characterization of Accessible Chromatin Regions in Cattle Rumen Epithelial Tissue during Weaning

“…TGFβ1 (transforming growth factor-β 1) is a secreted protein member of the transforming growth factor-β superfamily of cytokines that has several functions, including cell growth, proliferation and differentiation, and apoptosis [ 44 ]. Moreover, TGFβ1 can affect cell adhesion, according to studies conducted in humans, sheep, and cattle [ 45 , 46 , 47 ]. As previously reported in the cattle [ 48 ], during weaning, TGFβ1 was identified as a putative mediator of rumen epithelial tissue development.…”

Brisket Disease Is Associated with Lower Volatile Fatty Acid Production and Altered Rumen Microbiome in Holstein Heifers

“…The rumen plays an important role in providing necessary nutrients to the animal [ 8 ] and has several important physiological functions, including the absorption of volatile fatty acids (VFAs) [ 9 ], nutrient transport [ 10 ], and metabolic activity and protection [ 11 ]. VFAs are crucial to the maintenance, growth, and production performance of ruminants [ 12 ].…”