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. 2019 Jan 8;39(1):BSR20180270.
doi: 10.1042/BSR20180270. Print 2019 Jan 31.

Functional and Structural Studies of Tolloid-Like 1 Mutants Associated With Atrial-Septal Defect 6

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Free PMC article

Functional and Structural Studies of Tolloid-Like 1 Mutants Associated With Atrial-Septal Defect 6

Lukasz Sieron et al. Biosci Rep. .
Free PMC article

Abstract

Inactive mammalian tolloid-like 1 (tll1) and mutations detected in tolloid-like 1 (TLL1) have been linked to the lack of the heart septa formation in mice and to a similar human inborn condition called atrial-septal defect 6 (ASD6; OMIM 613087, formerly ASD II). Previously, we reported four point mutations in TLL1 found in approximately 20% of ASD6 patients. Three mutations in the coding sequence were M182L, V238A, and I629V. In this work, we present the effects of these mutations on TLL1 function. Three recombinant cDNA constructs carrying the mutations and one wild-type construct were prepared and then expressed in HT-1080 cells. Corresponding recombinant proteins were analyzed for their metalloendopeptidase activity using a native substrate, chordin. The results of these assays demonstrated that in comparison with the native TLL1, mutants cleaved chordin and procollagen I at significantly lower rates. CD analyses revealed significant structural differences between the higher order structure of wild-type and mutant variants. Moreover, biosensor-based assays of binding interactions between TLL1 variants and chordin demonstrated a significant decrease in the binding affinities of the mutated variants. The results from this work indicate that mutations detected in TLL1 of ASD6 patients altered its metalloendopeptidase activity, structure, and substrate-binding properties, thereby suggesting a possible pathomechanism of ASD6.

Keywords: atrial-septal defect; endopeptidase activity; mammalian tolloid-like 1; point mutation; protein structure; tolloid.

Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. Representative histograms of DNA sequencing of constructs with introduced mutations
The mutated nucleotides are framed.
Figure 2
Figure 2. Pattern of WB analysis of proteins secreted into the culture medium and retained inside of selected HT-1080 clones being stable producers of the recombinant TLL1 and its mutated variants
(A) Detection following Western blot of TLL1 in culture medium of HT-1080 cells that were transfected with plasmids and grown in the presence of G-418. Top picture – detection of TLL1s, bottom picture – detection of β actin. First five lanes from left contained proteins from culture medium of mock transfected HT-1080 (HT-1080), or with plasmid carrying cDNA encoding native TLL1 (WT) and TLL1 with mutations (M182L, V238A, I629V). Next four lanes represent proteins in lysates from the same cells. (B) Results of electrophoresis of equal volumes of purified recombinant proteins (40 ng/μl). Lanes contained; WT - native TLL1, M182L, V238A and I629V – mutated variants of TLL1, BSA - bovine serum albumin.
Figure 3
Figure 3. The comparison of migration of purified recombinant TLL1 enzymes following SDS/PAGE
Lanes from left: Molecular mass marker, two sets of four lanes each with unreduced samples or reduced samples as in Figure 2B. The asterisks refer to the mutant sample that migrated at a different rate than the other samples.
Figure 4
Figure 4. TLL1 enzymatic activity tests against synthetic labeled peptide, chordin, and procollagen I. The cleavage of chordin and procollagen were detected by electrophoresis of the reaction products following their incubation with recombinant wild-type or mutated variants of TLL1
(A) Lowered cleavage rates for the mutated variants of TLL1. The histograms represent percent of wild-type TLL1 activity on fluorescently labeled peptide MCA-YVADP-DNP-K – slashed filed boxes, chordin – doted boxes and procollagen I – black boxes. Statistically significant difference between wild-type and mutated variants are indicated by asterisks: *P<0.05, **P<0.01, ***P<0.001. (B) Results of cleavage of chordin: lanes from left contained reaction products of chordin incubated in the enzymes absence (Blank) or in the presence of: proteins obtained from culture medium of mock transfected HT-1080 cells (HT-1080), purified recombinant enzymes of wild-type (WT) or its mutated variants (M182L, V238A, I629V). (C) Pattern of electrophoretic separation of the cleavage products of procollagen I in the presence of wild type TLL1 and its mutated variants. Lanes contained: molecular weight marker (M) and procollagen I incubated without enzymes (0), and with proteins obtained from culture medium of mock transfected HT-1080 cells (HT-1080), purified recombinant enzymes of wild-type (WT) or its mutated variants (M182L, V238A, I629V). Abbreviations: pNα1(I) – α 1 chain with amino-terminal propeptide of collagen type I; pNα2(I) – α 2 chain with amino-terminal propeptide of collagen type I.
Figure 5
Figure 5. Percent of the polypeptide structure content in TLL1 and its mutants determined by CD spectrometry measurements
Empty boxes – TLL1, slashed boxes – M182L, hatched horizontally boxes – V238A, Filled boxes – I629V. The statistically significant differences (P<0.05 - “*”; P<0.01 - “**”; P<0.001 - “***”) in structures content between TLL1 and its mutated variants are indicated above the boxes.

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