Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies.

Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.

Metabolomic profiling to characterize acute intestinal ischemia/reperfusion injury.

Sepsis and septic shock are the leading causes of death in critically ill patients. Acute intestinal ischemia/reperfusion (AII/R) is an adaptive response to shock. The high mortality rate from AII/R is due to the severity of the disease and, more importantly, the failure of timely diagnosis. The objective of this investigation is to use nuclear magnetic resonance (NMR) analysis to characterize urine metabolomic profile of AII/R injury in a mouse model. Animals were exposed to sham, early (30 min) or late (60 min) acute intestinal ischemia by complete occlusion of the superior mesenteric artery, followed by 2 hrs of reperfusion. Urine was collected and analyzed by NMR spectroscopy. Urinary metabolite concentrations demonstrated that different profiles could be delineated based on the duration of the intestinal ischemia. Metabolites such as allantoin, creatinine, proline, and methylamine could be predictive of AII/R injury. Lactate, currently used for clinical diagnosis, was found not to significantly contribute to the classification model for either early or late ischemia. This study demonstrates that patterns of changes in urinary metabolites are effective at distinguishing AII/R progression in an animal model. This is a proof-of-concept study to further support examination of metabolites in the clinical diagnosis of intestinal ischemia reperfusion injury in patients. The discovery of a fingerprint metabolite profile of AII/R will be a major advancement in the diagnosis, treatment, and prevention of systemic injury in critically ill patients.

The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11.

Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function.

Pbx-dependent regulation of lbx gene expression in developing zebrafish embryos.

Ladybird (Lbx) homeodomain transcription factors function in neural and muscle development--roles conserved from Drosophila to vertebrates. Lbx expression in mice specifies neural cell types, including dorsally located interneurons and association neurons, within the neural tube. Little, however, is known about the regulation of vertebrate lbx family genes. Here we describe the expression pattern of three zebrafish ladybird genes via mRNA in situ hybridization. Zebrafish lbx genes are expressed in distinct but overlapping regions within the developing neural tube, with strong expression within the hindbrain and spinal cord. The Hox family of transcription factors, in cooperation with cofactors such as Pbx and Meis, regulate hindbrain segmentation during embryogenesis. We have identified a novel regulatory interaction in which lbx1 genes are strongly downregulated in Pbx-depleted embryos. Further, we have produced a transgenic zebrafish line expressing dTomato and EGFP under the control of an lbx1b enhancer--a useful tool to acertain neuron location, migration, and morphology. Using this transgenic strain, we have identified a minimal neural lbx1b enhancer that contains key regulatory elements for expression of this transcription factor.

Expression of two insm1-like genes in the developing zebrafish nervous system.

Insulinoma associated-1 (INSM1, formerly IA-1) is a Cys(2)-His(2) zinc finger transcription factor sharing conserved regions with Caenorhabditis elegans EGL-46 and Drosophila Nerfin-1. INSM, EGL-46, and Nerfin proteins comprise the EIN family of zinc finger transcription factors. egl-46 and nerfin-1 have been implicated in various aspects of neuronal differentiation including cell fate specification, axon guidance decisions and cell migration. Murine Insm1 has a restricted expression pattern in the developing CNS. We have characterized two zebrafish (Danio rerio) Insm1-like genes, insm1a and insm1b, and analyzed their expression patterns during embryonic development. Zebrafish insm1a and insm1b share an embryonic expression pattern comparable to the proneural deltaA as well as overlapping the neuronal marker elavl3. The expression pattern observed for zebrafish insm1a and insm1b is similar to other EIN homologues. Both zebrafish insm1-like transcripts are also present in a region of the embryo where pancreatic progenitors originate. The expression data along with functional characterization of invertebrate homologues suggest a conserved pathway involving the EIN transcription factors in early neurogenesis.