Did Androgen-Binding Protein Paralogs Undergo Neo- and/or Subfunctionalization as the Abp Gene Region Expanded in the Mouse Genome?

Abstract

The Androgen-binding protein (Abp) region of the mouse genome contains 30 Abpa genes encoding alpha subunits and 34 Abpbg genes encoding betagamma subunits, their products forming dimers composed of an alpha and a betagamma subunit. We endeavored to determine how many Abp genes are expressed as proteins in tears and saliva, and as transcripts in the exocrine glands producing them. Using standard PCR, we amplified Abp transcripts from cDNA libraries of C57BL/6 mice and found fifteen Abp gene transcripts in the lacrimal gland and five in the submandibular gland. Proteomic analyses identified proteins corresponding to eleven of the lacrimal gland transcripts, all of them different from the three salivary ABPs reported previously. Our qPCR results showed that five of the six transcripts that lacked corresponding proteins are expressed at very low levels compared to those transcripts with proteins. We found 1) no overlap in the repertoires of expressed Abp paralogs in lacrimal gland/tears and salivary glands/saliva; 2) substantial sex-limited expression of lacrimal gland/tear expressed-paralogs in males but no sex-limited expression in females; and 3) that the lacrimal gland/tear expressed-paralogs are found exclusively in ancestral clades 1, 2 and 3 of the five clades described previously while the salivary glands/saliva expressed-paralogs are found only in clade 5. The number of instances of extremely low levels of transcription without corresponding protein production in paralogs specific to tears and saliva suggested the role of subfunctionalization, a derived condition wherein genes that may have been expressed highly in both glands ancestrally were down-regulated subsequent to duplication. Thus, evidence for subfunctionalization can be seen in our data and we argue that the partitioning of paralog expression between lacrimal and salivary glands that we report here occurred as the result of adaptive evolution.

Studies of an Androgen-Binding Protein Knockout Corroborate a Role for Salivary ABP in Mouse Communication

“…RNA isolation and complementary DNA (cDNA) library synthesis was performed as previously detailed (Karn et al 2014). qPCR was performed on an ABI Prism 7000 machine using the Maxima SYBR green qPCR kit from Thermo Scientific (Karn et al 2014).…”

“…qPCR was performed on an ABI Prism 7000 machine using the Maxima SYBR green qPCR kit from Thermo Scientific (Karn et al 2014). We generated a standard curve for each primer using male submandibular B6 cDNA templates for three Abp paralog primers (a27, bg26, and bg27) and the remaining standard curves were produced from male lacrimal cDNA template (ABI qPCR manual).…”

“…We calculated the expression of Abp transcripts in each genotype using a relative quantification analysis, where each sample was compared using the standard curves generated for each primer set amplified by the B6 genome mouse cDNA library, described in detail in File S1 (Karn et al 2014). The ANOVA statistical test was performed to determine interactions between genotype and gender for both salivary and lacrimal glands, and genotype and transcript expression levels in the salivary glands.…”

“…The samples were provided to the Proteomics Core Facility of the University of Arizona where personnel digested 2 mg of each with trypsin. Liquid chromatographymass spectrometry/mass spectrometry analysis of the digested proteins in the gel pieces (Shevchenko et al 1996) was carried out using an LTQ Orbitrap Velos mass spectrometer described in Karn et al (2014) (File S1). Peptides were eluted onto an analytical column and separated using gradients composed of acetonitrile and formic acid.…”

“…Before analyzing protein and transcript expression in the three genotypes, we performed a general evaluation of tissue structure in the lacrimal and salivary glands (parotid, submandibular, and sublingual) that are known to be major sites of Abp gene expression (Dlouhy et al 1986;Karn et al 2014). In addition, we analyzed the Harderian gland (the source of heteroglobin that may mediate behavior in hamsters) (Dominguez 1995;Alvarez et al 2002); kidney, which was compromised in a uteroglobin (Ug) knockout (Zhang et al 1997); and liver, a major site of detoxification that might be affected by toxins that could not be bound and removed in an Abp knockout animal (Zhou et al 2011).…”