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Comparative Study
. 2012 Jan 31;53(1):461-9.
doi: 10.1167/iovs.10-5955.

ADAMTSL4, a secreted glycoprotein widely distributed in the eye, binds fibrillin-1 microfibrils and accelerates microfibril biogenesis

Luis A R Gabriel  1 , Lauren W Wang, Hannah Bader, Jason C Ho, Alana K Majors, Joe G Hollyfield, Elias I Traboulsi, Suneel S Apte
Affiliations
Comparative Study

ADAMTSL4, a secreted glycoprotein widely distributed in the eye, binds fibrillin-1 microfibrils and accelerates microfibril biogenesis

Luis A R Gabriel et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: ADAMTSL4 mutations cause autosomal recessive isolated ectopia lentis (IEL) and ectopia lentis et pupillae. Dominant FBN1 mutations cause IEL or syndromic ectopia lentis (Marfan syndrome and Weill-Marchesani syndrome). The authors sought to characterize recombinant ADAMTSL4 and the ocular distribution of ADAMTSL4 and to investigate whether ADAMTSL4 influences the biogenesis of fibrillin-1 microfibrils, which compose the zonule.

Methods: ADAMTSL4 was expressed by the transfection of HEK293F cells. Protein extracts and paraffin sections from human eyes were analyzed by Western blot analysis and by immunoperoxidase staining, respectively. Immunofluorescence was used to evaluate fibrillin-1 deposition in the ECM of fetal bovine nuchal ligament cells after culture in ADAMTSL4-conditioned medium or control medium. Confocal microscopy was performed to investigate ADAMTSL4 and fibrillin-1 colocalization in these cultures.

Results: Western blot analysis identified ADAMTSL4 as a glycoprotein in HEK293F cells and as a major band of 150 kDa in ocular tissues including ciliary body, sclera, cornea, and retina. Immunoperoxidase staining showed a broad ocular distribution of ADAMTSL4, associated with both cells and fibrillar ECM. When cultured in ADAMTSL4-containing medium, fetal bovine nuchal ligament cells showed accelerated fibrillin-1 deposition in ECM. ADAMTSL4 colocalized with fibrillin-1 microfibrils in the ECM of these cells.

Conclusions: ADAMTSL4 is a secreted glycoprotein that is widely distributed in the human eye. Enhanced fibrillin-1 deposition in the presence of ADAMTSL4 and colocalization of ADAMTSL4 with fibrillin-1 in the ECM of cultured fibroblasts suggest a potential role for ADAMTSL4 in the formation or maintenance of the zonule.

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Figures

Figure 1.
Figure 1.
Western blot of recombinant ADAMTSL4. (A) Analysis of medium from HEK293F cells stably transfected with ADAMTSL4 or empty vector shows detectable ADAMTSL4 protein only in ADAMTSL4-transfected cells. Solid arrow: major 140- to 150-kDa species; asterisks: presumed proteolytic fragments. The identities of the 250-kDa species marked by the arrowhead and the species indicated by the broken arrow are unknown, although these species are also attenuated by preblocking the antibody with immunogen peptides (Supplementary Fig. S1A, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-5955/-/DCSupplemental). (B) Enzymatic deglycosylation of ADAMTSL4 in conditioned medium of stably transfected HEK293F cells was performed using peptide N-glycosidase F. Increased electrophoretic mobility of anti-ADAMTSL4 reactive species is seen in the presence of peptide N-glycosidase F (+) compared with the untreated control (−). (C) Enzymatic deglycosylation of ADAMTSL4 in conditioned medium of stably transfected HEK293F cells was performed using O-glycosidase (+). Increased electrophoretic mobility of anti-ADAMTSL4 reactive species is seen after O-glycosidase treatment.
Figure 2.
Figure 2.
Immunolocalization of ADAMTSL4 in transiently transfected COS-1 cells. Punctate distribution in (A) ECM of nonpermeabilized cells and (B) cytoplasmic staining in permeabilized cells. (A) The plane of focus in (A) is the substratum underneath the imaged cells. The center row demonstrates staining in the subcellular ECM “footprint” left behind by a detached cell; hence, no DAPI stained nuclei are seen here. As a positive control for the staining procedure, and as a representative member of the ADAMTSL family, a punctate staining pattern in ECM was seen in myc-His6–tagged ADAMTSL1-transfected cells immunostained with anti-myc (C, left). Absence of staining in cells where the primary antibody was omitted (C, right). Scale bars, 50 μm.
Figure 3.
Figure 3.
Western blot analysis of various components dissected from a normal human eye. The source for each sample is indicated above the corresponding lanes. The molecular weight markers (in kDa) are shown at the left of each panel. cb, ciliary body; rs, retinal tissue.
Figure 4.
Figure 4.
Localization of ADAMTSL4 in the anterior segment of a normal human eye. ADAMTSL4 signal is brown, and the section is counterstained with hematoxylin (blue). (A′), (B′), (D′) and (F′) are negative control images corresponding to (A), (B), (D) and (E) were obtained from sections in which the primary antibody was omitted. (A) Cornea. Epithelium (E) and endothelium (En) are indicated. (B) Iris stroma. Signal is primarily seen in fibrillar ECM. (C) Trabecular meshwork (TM). Asterisk: canal of Schlemm. Co, cornea; I, iris. (D) Ciliary body. The unpigmented epithelium is indicated by an arrow. Note that the antibody signal is primarily within the ECM of the ciliary body (see control D′ and Supplementary Fig. S1B, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-5955/-/DCSupplemental). (E) Lens shows immunostaining in the lens cortex in the equatorial region. LE, lens epithelium. Scale bars, 50 μm.
Figure 5.
Figure 5.
Localization of ADAMTSL4 in the posterior segment of a normal human eye. ADAMTSL4 signal is brown, and the sections are counterstained with hematoxylin (blue). (A′–E′) From sections stained without primary antibody as a negative control. (A–E) Stained sections. (A) Retina. OFL, optic fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer, RPE, retinal pigment epithelium. (B) Ch, choroid; Sc, sclera. (C) Sclera. (D) Optic nerve. Note ADAMTSL4 staining in ECM of sclera, choroid, and optic nerve. (E) Retinal blood vessel. Arrows: cellular staining in vascular smooth muscle cells. Scale bars, 50 μm.
Figure 6.
Figure 6.
ADAMTSL4 accelerates fibrillin-1 deposition in the matrix of cultured fibroblasts. Fetal bovine nuchal ligament cells (fBNL) cells were cultured to confluence in media that had previously been conditioned by HEK293F cells stably transfected with ADAMTSL4 expression plasmid or empty vector as indicated. After 4 days of culture, cells were fixed and stained for fibrillin-1 (green), and nuclei were stained with DAPI (blue). Three representative fields (20× magnification) from independent experiments are shown for both vector-conditioned and ADAMTSL4-conditioned medium–treated fBNL cells. Note the enhanced fibrillin-1 fluorescence in cells cultured in the presence of exogenous ADAMTSL4. Scale bars, 50 μm.
Figure 7.
Figure 7.
ADAMTSL4 colocalizes with fibrillin-1 microfibrils in ECM of cultured fibroblasts. Combined immunofluorescence for ADAMTSL4 (red) and fibrillin-1 (green) was performed after culture of fBNL cells in the presence of ADAMTSL4-conditioned medium or without the addition of conditioned medium. (A) Confocal microscopy. In ADAMTSL4 medium–treated cells, confocal images show that ADAMTSL4 distribution overlaps with fibrillin-1 (yellow, merged image). (B) Conventional fluorescence microscopy. Cells were incubated with vector-conditioned medium. ADAMTSL4 was also detected in these cultures and colocalized with fibrillin-1 immunostaining (yellow), suggesting the production of ADAMTSL4 by fBNL cells or HEK293F cells. (C) fBNL cultures were grown without the addition of any conditioned medium. Note the presence of ADAMTSL4 on fibrillin-1 microfibrils, indicating the production of ADAMTSL4 by fBNL cells (see Supplementary Fig. S2, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-5955/-/DCSupplemental). Scale bars, 50 μm.
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