The involvement of proline-rich protein Mus musculus predicted gene 4736 in ocular surface functions

Abstract

Aim: To research the two homologous predicted proline-rich protein genes, Mus musculus predicted gene 4736 (MP4) and proline-rich protein BstNI subfamily 1 (Prb1) which were significantly upregulated in cultured corneal organs when encountering fungal pathogen preparations. This study was to confirm the expression and potential functions of these two genes in ocular surface.

Methods: A Pseudomonas aeruginosa keratitis model was established in Balb/c mice. One day post infection, mRNA level of MP4 was measured using real-time polymerase chain reaction (PCR), and MP4 protein detected by immunohistochemistry (IHC) or Western blot using a customized polyclonal anti-MP4 antibody preparation. Lacrimal glands from normal mice were also subjected to IHC staining for MP4. An online bioinformatics program, BioGPS, was utilized to screen public data to determine other potential locations of MP4.

Results: One day after keratitis induction, MP4 was upregulated in the corneas at both mRNA level as measured using real-time PCR and protein levels as measured using Western blot and IHC. BioGPS analysis of public data suggested that the MP4 gene was most abundantly expressed in the lacrimal glands, and IHC revealed that normal murine lacrimal glands were positive for MP4 staining.

Conclusion: MP4 and Prb1 are closely related with the physiology and pathological processes of the ocular surface. Considering the significance of ocular surface abnormalities like dry eye, we propose that MP4 and Prb1 contribute to homeostasis of ocular surface, and deserve more extensive functional and disease correlation studies.

Keywords: Mus musculus predicted gene 4736; Pseudomonas aeruginosa keratitis; ocular surface; proline-rich protein.

Figure 1. Homology of MP4 (NP_444481) and Prb1 (NP_941071) proteins as revealed by BLAST analysis.

Figure 2. Expression of MP4 in murine corneas

PaK was established in mice. One day later, corneas were harvested for mRNA (A) or protein extraction (B), or subjected to HE or gram staining or IHC (C). In the latter case, normal rabbit serum was used as substitute for the anti-MP4 antibody to validate the specificity of the primary antibody, and the results are shown as “negative” staining control. In A and B, three corneas from PaK models or normal control were pooled as one sample respectively. All experiments were conducted at least twice.

Figure 3. Preferential expression of MP4 in lacrimal glands as revealed by BioGPS

Expression intensity was given by BioGPS. “All other tissues” represents the tissues or cells that were included in BioGPS but not detailed here, including white adipose, adrenal gland, amygdala, bladder, bone, bone marrow, cerebellum, cerebral cortex, ciliary bodies, cornea, dorsal root ganglia, dorsal striatum, epidermis, hippocampus, lung, lymph nodes, lactating mammary gland, non-lactating mammary gland, nucleus accumbens, olfactory bulb, ovary, Pancreas, pituitary, prostate, retina, skeletal muscle, spinal cord, spleen, stomach, umbilical cord, small intestine, uterus, and large intestine.

Figure 4. Immunostaining of MP4 in normal mouse lacrimal glands

Sections were subjected to HE staining and immunostaining respectively. Parallel handling with normal rabbit serum but without anti-MP4 antibodies was run as “negative” control.