QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00850

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bhattacharya, G. Articles by Cosgrove, D. Search for Related Content PubMed PubMed Citation Articles by Bhattacharya, G. Articles by Cosgrove, D. Social Bookmarking                         What's this?

A domain-specific usherin/collagen IV interaction may be required for stable integration into the basement membrane superstructure

¹ Usher Syndrome Center, Boys Town National Research Hospital, 555 No. 30^th Street, Omaha, Nebraska, 68131, USA
² Program in Matrix Biology, Gastroenterology and Renal Divisions, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA

View larger version (11K): [in a new window]   Fig. 2. Major structural elements of the usherin protein based on amino acid sequence. The amino acid positions where domains start and end are indicated. The location of peptides used to derive antibodies 1 and 2 used in these studies are shown. Constructs used to generate fusion peptides comprised the indicated portions of the thrombospondin 1, LN, LE and fibronectin type III domains (Ts-FP, LN-FP, LE-FP, and FNFP, respectively).

View larger version (56K): [in a new window]   Fig. 1. Usherin colocalizes with type IV collagen. (A) A mid-modiolar cross section of murine cochlea was subjected to dual immunofluorescence staining using anti-usherin and anti-type IV collagen-specific antibodies. (B) Immunogold colocalization of usherin and type IV collagen. Testis was embedded in Unicryl embedding resin and ultrathin sections were reacted with antibodies specific for either usherin (rabbit anti-usherin) or type IV collagen (goat anti-collagen IV). Sections were then reacted with both 10 nm gold-conjugated anti-rabbit secondary antibodies and 5 nm gold-conjugated anti-goat secondary antibodies. Arrows denote typically observed colocalization for these two antibody probes. Scale bar: A, 50 µm; B, 0.2 µm.

View larger version (22K): [in a new window]   Fig. 3. The usherin fusion peptides appear to be properly folded. (A) Usherin fusion peptides were fractionated by PAGE on 15% SDS (denatured) or non-SDS (non-denatured) gels under reducing or non-reducing conditions. (B) Five micrograms of recombinant usherin (ush) or the indicated peptides were analyzed for free sulfhydryl groups under reducing and non-reducing conditions using the Thiol and Sulfide Quantitation Kit (Molecular Probes, Eugene, OR). Free sulfhydryls result in the formation of a colored product with an absorption maxima at 410 nm, which is quantified spectrophotometrically.

View larger version (60K): [in a new window]   Fig. 4. The LE domain of usherin interacts with type IV collagen. Extracts of matrix from the indicated mouse tissues were (A) reacted with the fusion peptide consisting of the LE domain, immunoprecipitated with anti-GST antibodies, and the immunoprecipitate western blotted using anti-type IV collagen antibodies, or (B) directly immunoprecipitated with anti-type IV collagen antibodies and the immunoprecipitate western blotted using anti-usherin antibodies. The molecular mass markers are given in kilodaltons.

View larger version (18K): [in a new window]   Fig. 5. The LE domain of usherin interacts with the 7S domain and the triple helical domain of type IV collagen. Direct ELISA determined interaction between the domain-specific usherin fusion peptides and the indicated domains of type IV collagen. Fusion peptides were reacted with purified collagen domains, which were immobilized on plastic microtiter dishes. Standard deviations are given for triplicate measurements. TIV protomer, the full-length trimeric protomer of type IV collagen; TH, the triple helical domain of type IV collagen; NC1, the non-collagenous domain of type IV collagen.

View larger version (17K): [in a new window]   Fig. 6. Binding of usherin to type IV collagen. Binding was analyzed by coupling usherin to a sensor chip and reacting with the indicated concentration range of type IV collagen. Response is measured in resonance units (RU).

View larger version (80K): [in a new window]   Fig. 7. (A) Expression and purification of full-length recombinant human and mouse usherin protein. The full-length cDNAs for human and mouse usherin were cloned into pcDNA 3.1 and transfected into 293 cells. Clones secreting the highest amounts were grown, the supernatant concentrated and recombinant usherin purified by affinity chromatography over nickel columns. Samples of the purified protein were subjected to SDS-PAGE, and the protein stained with Coomassie Blue. The arrow indicates stained protein of the predicted molecular size for usherin (175 kDa). (B) Competitive inhibition of immunostaining using recombinant usherin protein. (Top) Immunostaining with antibody 2 (see Fig. 1). (Bottom) The antibody was mixed with 100 ng of recombinant usherin protein prior to applying it on the tissue section. Absence of staining in B proves that the recombinant protein shown in A is indeed usherin. Both mouse and human usherin competed equally well.

View larger version (35K): [in a new window]   Fig. 8. Missense mutations in the b loop of the first and fourth LE domains abolish the usherin/collagen interaction. (A) Purified type IV collagen and recombinant human usherin (1 µg of each) were mixed with varying relative molar excess amounts of LE domain fusion peptide. Following incubation, complexes were immunoprecipitated with anti-collagen (IV) antibodies, and the immunoprecipitated material digested with collagenase. Remaining material was fractionated by PAGE, and stained with Coomassie Blue. (B) (Left) The three-dimensional structure of a single LE module, based on the mouse laminin chain crystal structure. Substituted amino acid residues that obliterate type IV collagen binding activity are shown in red, and residues that did not affect binding are in green. (Right) Location of missense mutations in the LE repeats of usherin. Mutations that abolish collagen binding are in red; mutations that do not affect collagen binding are in green. Mutations in black were not tested.

View larger version (25K): [in a new window]   Fig. 9. Usherin binding to type IV collagen may facilitate stable integration of usherin in the basement membrane. (A) Basal lamina was extracted from normal (C) and Alport (A) testes. Solubilized proteins were subjected to immunoprecipitation with anti-usherin antibodies and a western blot probed with anti-type IV collagen 3, 4 and 5 antibodies (lanes 1 and 2); immunoprecipitated with anti-type IV collagen 1 and 2 antibodies and a western blot probed with anti-usherin antibodies (lanes 3 and 4); or the crude extract analyzed directly (by western blot and probed with anti-usherin antibodies (lanes 5 and 6). Molecular mass markers are in kilodaltons. (B) The experiment in A was run in triplicate using three different sets of animals, and the chemiluminescence signal was quantified and analyzed statistically as described in Materials and Methods. The quantitative data shown in B correspond to the lanes in A.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter