Usherin expression is highly conserved in mouse and human tissues

doi:10.1016/S0378-5955(02)00635-4

Hearing Research

Volume 174, Issues 1–2, December 2002, Pages 55-63

https://doi.org/10.1016/S0378-5955(02)00635-4 Get rights and content

Abstract

Usher syndrome is an autosomal recessive disease that results in varying degrees of hearing loss and retinitis pigmentosa. Three types of Usher syndrome (I, II, and III) have been identified clinically with Usher type II being the most common of the three types. Usher type II has been localized to three different chromosomes 1q41, 3p, and 5q, corresponding to Usher type 2A, 2B, and 2C respectively. Usherin is a basement membrane protein encoded by the USH2A gene. Expression of usherin has been localized in the basement membrane of several tissues, however it is not ubiquitous. Immunohistochemistry detected usherin in the following human tissues: retina, cochlea, small and large intestine, pancreas, bladder, prostate, esophagus, trachea, thymus, salivary glands, placenta, ovary, fallopian tube, uterus, and testis. Usherin was absent in many other tissues such as heart, lung, liver, kidney, and brain. This distribution is consistent with the usherin distribution seen in the mouse. Conservation of usherin is also seen at the nucleotide and amino acid level when comparing the mouse and human gene sequences. Evolutionary conservation of usherin expression at the molecular level and in tissues unaffected by Usher 2a supports the important structural and functional role this protein plays in the human. In addition, we believe that these results could lead to a diagnostic procedure for the detection of Usher syndrome and those who carry an USH2A mutation.

Introduction

Out of the 20 000 deaf and blind people in the United States it is estimated that over half have Usher syndrome. Usher syndrome is an autosomal recessive disease characterized by varying degrees of hearing loss and retinitis pigmentosa (RP). Three types of Usher syndrome (I, II, and III) have been identified clinically and are distinguished by severity and progression of hearing loss along with the presence or absence of vestibular dysfunction. Usher is clinically and genetically heterogeneous. To date, 12 loci and six genes have been identified (Eudy et al., 1998, Kimberling et al., 1990, Kimberling et al., 1995, Pieke-Dahl et al., 2000, Smith et al., 1992, Ahmed et al., 2001, Petit, 2001, Sankila et al., 1995). The frequency of Usher has been estimated at 4.4/100 000 in the US (Boughman et al., 1983) and 3.0/100 000 in Scandinavia (Hallgren, 1959).

Usher type II is the most frequent type of Usher, comprising over half of all cases (Rosenberg et al., 1997). Usher II is a mild form of Usher syndrome and most cases are caused by mutations in the USH2A gene. Moderate to severe sensorineural hearing impairment at birth and progressive RP clinically characterize the syndrome. A profound hearing loss and vestibular problems distinguish Usher I from Usher II. Linkage studies have subdivided Usher II into three molecular subtypes: Usher IIA, IIB, and IIC which localize to chromosomes 1q41, 3p, and 5q respectively (Pieke-Dahl et al., 2000, Hmani et al., 1999, Kimberling et al., 1991). The USH2A gene encodes a novel protein, usherin, found in the basement membrane of several murine tissues (Bhattacharya et al., 2002). Usherin is a large glycoprotein with an approximate molecular weight of 171.5 kDa. It contains four domains (thrombospondin, laminin type IV, laminin epidermal growth factor (EGF)-like, and fibronectin type III) common among protein components of the basal lamina and extracellular matrices (Weston et al., 2000). The structure of the usherin protein is displayed in Fig. 1. The domain nearest the amino terminal is a 300 amino acid sequence with greatest homology to thrombospondin type 1. The laminin-like domain (LN) spans the next 200 amino acids in the protein. This region is followed by a stretch of 10 laminin EGF-like (LE) repeats that comprise 500 amino acids. The LE repeats are rod-like laminin EGF modules arranged in series (Bhattacharya et al., 2002, Bork et al., 1996, Beck et al., 1990). At the carboxy terminus, there are four repeats, 100 amino acids each, with structural homology to fibronectin type III domains. The structural and functional importance of usherin has been confirmed through mutation studies, which demonstrate that a pathologic mutation in any of the usherin domains can result in an Usher IIa phenotype (Weston et al., 2000).

This study aims to investigate the distribution of usherin in human tissues. In addition, the conservation of usherin expression between mouse and human tissues is assessed and demonstrates that usherin expression is highly conserved between the two species.

Section snippets

Gene homology analysis

The NCBI BLAST program was used to align the human and mouse USH2a nucleotide and amino acid sequences.

LN domain expression and antibody production

The cDNA sequence encoding the LN domain of human usherin was cloned into the FLAG-ATS expression system (Sigma, St. Louis, MO, USA), expressed in Escherichia coli, and recombinant protein was purified by nickel affinity chromatography according to the manufacturers instructions. The peptide was run on a 12% polyacrylamide gel along with molecular standards. The gel was stained with Coomassie

Specificity of the antibody

The purity of the cloned LN domain used to raise the usherin antibody is demonstrated in Fig. 2A. A single band was detected using a Coomassie assay at the correct molecular size predicted for the LN domain.

A Western blot was used to test the specificity of the usherin antibody used in this study. In Fig. 2B, protein extracts from leukocytes, placenta, and purified recombinant usherin protein were probed with the usherin antibody. A single band was detected at the appropriate molecular size

Discussion

Immunoperoxidase antibody staining demonstrates that usherin is localized in structural and vascular basement membranes of several human tissues, however its presence is not ubiquitous. Usherin is seen in the basement membranes of smooth muscle, glandular tissue, vasculature, and peripheral nerves.

The location of usherin in human tissues directly parallels its location in the mouse, indicating that the regulation of tissue specific expression for this protein has been evolutionary conserved.

Acknowledgements

This work was supported by a grant from the NIH-NIDCD (P01-DC01813-08) and a grant from the Foundation for Fighting Blindness, USA. We would also like to thank Monica M. Jablonski at University of Tennessee for her contribution of human eye tissue to this research.

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¹: These authors contributed equally to this work.

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Usherin expression is highly conserved in mouse and human tissues

Abstract

Introduction

Section snippets

Gene homology analysis

LN domain expression and antibody production

Specificity of the antibody

Discussion

Acknowledgements

Am. J. Hum. Genet.

Hear. Res.

J. Chronic Dis.

Genomics

J. Biol. Chem.

J. Biol. Chem.

Genomics

Kidney Int.

Genomics

Am. J. Hum. Genet.

Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains

J. Histochem. Cytochem.