Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families

Abstract

The extracellular matrix (ECM) regulates numerous cellular events in addition to providing structural integrity. Among several protein and enzyme families implicated in functions of the ECM, the lysyl oxidases and ADAMTS proteins are known to participate in microfibril and elastic fiber formation as well as ECM-associated signaling. A yeast two-hybrid screen to identify lysyl oxidase (LOX) binding proteins identified ADAMTSL4 as a potential interactor. We demonstrate here that several members of the LOX and ADAMTS families interact with one another. Upon investigating the interaction between LOX and ADAMTSL2 we found that the absence or inhibition of Lox affected ADAMTSL2 molecular forms and reduced its tissue levels. Thus, ADAMTSL2 stability and inter-molecular complexes may depend on the activity of lysyl oxidases.

Figure 1. LOX complexes with ADAMTSL4

A. A yeast-two-hybrid (Y2H) screen using LOX as the bait identified three distinct ADAMTSL4 clones (clones 1-3). The selective interaction domain (SID) indicates the consensus interacting region of ADAMTSL4 determined by the overlap of the clones. B. Diploid yeast cells containing both a bait vector (LOX) and prey vector (ADAMTSL4 clone 1) were grown on nutrient permissive or selective plates along with negative controls of empty bait vector, empty prey vector and empty bait and prey vectors. The left-hand panel represents yeast colonies grown on (-)Leu (-)Trp permissive medium to maintain the growth of yeast containing both vectors, while the right-hand panel shows a replicate of the same plate on selective medium. Note that only colonies expressing both LOX and ADAMTSL4 grow on selective medium demonstrating that the two proteins interact. C. Autoradiograph of ³⁵S-labeled proteins transcribed in the TNT system. Co-immunoprecipitation of lysates expressing Lox plus ADAMTSL4 or Lox plus P85. A band corresponding to Lox is observed slightly under 50 kDa (bottom panel), while that corresponding for ADAMTSL4 is slightly under 150 kDa (top panel). P85 has a molecular mass of 85 kDa. In the TNT reactions where both proteins were translated (as observed in the input lane, right) bands corresponding to both ADAMTSL4 and LOX are observed upon pull down of either one of the proteins. In contrast, P85, which was co-translated with LOX (right input lane), is not observed following LOX pulldown.

Figure 2. LOX forms a complex with ADAMTSL2 and ADAMTS10

A. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing LOX plus ADAMTSL2, ADAMTSL2 alone (negative control), LOX plus ADAMTS10, or ADAMTS10 alone (negative control). B. Co-immunoprecipitation of stably transfected HEK293 cells expressing LOX plus ADAMTSL2 or LOX plus ADAMTS10 or parental HEK293 lysate (negative control). LOX migrates as two bands at ~50 kDa. ADAMTSL2 at ~150 kDa, and ADAMTS10 at 150 kDa. Membranes (A,B) were incubated with anti-myc (top) and anti-V5 (bottom). C. Proximity ligation assays performed on HEK293 cells expressing LOX+ADAMTSL2 (I, I′ IV, IV′), LOX+ADAMTS10 (II, II′) and LOX+P85 (III, III′). Anti-LOX and anti-myc antibodies were used to target LOX and ADAMTSL2/ADAMTS10 or P85, respectively. Signal amplification, marked by red signal (top, I-IV) or shown in grayscale (bottom, I′–IV′) is observed only in cells expressing LOX and an ADAMTS protein. In panel IV (control), no primary antibodies we added.

Figure 3. ADAMTSL2 and ADAMTS10 interact with LOXL2 and LOXL3

A,B. Co-immunoprecipitation from 4 day conditioned medium (A) or the cell lysate (B) from HEK293 cells stably transfected with ADAMTSL2 plus LOXL3 or LOXL3 alone. C. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing either LOXL2 plus ADAMTSL2, LOXL2 plus ADAMTS10, or LOXL2 alone.

Figure 4. ADAMTSL2 content is significantly reduced in mouse Lox^−/− tissues

A. Western blot for ADAMTSL2 from wt and Adamtsl2−/− embryos demonstrating ADAMTSL2 antibody specificity (A). B. Western blot analysis from E18.5 wt and Lox^−/− mouse eye (B), limb (C), lung (D) and brain (E) lysates. Distinct ADAMTSL2 protein bands, may arise from different glycosylation and/or processing in the different tissues. Molecular mass markers are shown on the left. Quantification of ADAMTSL2 levels measured from western blots (n=4 for each genotype) are shown to the right of each blot. Membranes were incubated with anti-ADAMTSL2 and anti-P97 as a control. * and *** indicate p< 0.05 and p<0.001, respectively.

Figure 5. βAPN treated mice show reduced ADAMTSL2 protein content

A. Western blot of pooled adult mouse aorta lysate from control and βAPN treated mice using anti-LOX. B. Quantification of LOX levels from control and βAPN treated mouse aortas from western blots (n=5; each lane is a pool of 6 aortas). LOX content is significantly upregulated following βAPN treatment. C. Western blot of pooled adult mouse aorta lysate from control and βAPN treated mice using ADAMTSL2. D. Quantification of ADAMTSL2 levels from control and βAPN treated mouse aortas from western blots (n=3; each lane is a pool of 6 aortas). ADAMTSL2 protein is significantly reduced following βAPN treatment. *** indicates p<0.001. M indicates molecular weight marker.

Figure 6. Alterations in putative ADAMTSL2 complexes following Lox deletion or inhibition

Upper parts of the gels shown in Figs. 4E and 5. A. ADAMTSL2 western blot from E18.5 wild-type and Lox^−/− brain lysates. B. Western blot of ADAMTSL2 in adult control and βAPN-treated mouse aorta lysates. Arrows mark high molecular weight bands which are affected (black) or not affected (grey), by the loss of Lox (A) or βAPN treatment (B). Membranes were incubated with anti-ADAMTSL2. M designates molecular weight marker.