Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors

Hyaline Fibromatosis Syndrome (HFS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype–phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized Ig-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Detailed medical information was not available for Patient 2. The patient showed signs of disease at birth and underwent long-term intensive physiotherapy as well as multiple operations for nodule removal. The patient is currently 16 and exhibits the classical signs of HFS, but was never subject to recurrent diarrhea or pulmonary infection.
Patient 3, a six week-old boy, was born five weeks preterm as the second child of consanguineous Turkish parents after an otherwise unremarkable pregnancy and delivery. Parameters for length and weight were within normal limits for gestational age and ethnicity. All other members of the family were healthy, including a three year-old sister. Initial reason for referral was a progressive painful limitation of joint mobility beginning in the first month of life. At that time, clinical examination revealed skin disease with thickening over the joints and hyperpigmentation on the areas overlying knuckles and malleoli. In the following months, pink nodules and plaques appeared on the face and extended to ears and neck; trunk, thighs and the perianal area were also affected by six months of age. Repeat examination showed generalized progressive contractures with characteristic 'frog-leg position', muscle wasting, and loss of subcutaneous fat. Motor milestones were delayed, but mental development was normal. Continuous morphine therapy was required for every-day handling of the child and to enable physiotherapy. Gingival hypertrophy caused difficulty during feeding. Failure to thrive was also due to recurrent episodes of diarrhea starting in the second month of life, often associated with prolonged fever and signs of bacterial infection. In spite of early and adequate therapeutic intervention, circulatory failure ensued during one infectious episode and the boy died at the age of 8 months.
Patient 4 is a 3 year-old boy, the second child of non-consanguineous Polish parents, born at term after an uneventful pregnancy. During the maternal ultrasound examination at 32 weeks of pregnancy, shorter tubular bones were suspected, but not confirmed at birth. Delivery occurred by caesarean section because of oligohydroamnios. Birth weight was 2800 g. During the first 5 weeks, the boy was treated because of talipes equinovarus. Progressive painful limitation of joint mobility began in first month of life. The baby did not tolerate physiotherapy (constant crying).
At the age of 5 months, he was referred to Neurology with a tentative diagnosis of arthrogryposis. Because of a slightly coarse face, macrocephaly (head circumference at 7 months of age was 47 cm, +2,46 SD) and limited joint mobility, the patient was strongly suspected of a storage disorder, which were ruled out by repeated enzymatic examinations. At the age of 10 months, the patient was referred to the Metabolic department, were "primary connective tissue disorder" was suspected. Xray analysis of bones suggested a diagnosis of infantile systemic hyalinosis. Because of exaggeration of protracted diarrhea, colonoscopy and gastroscopy were performed and lymphangiectasies were found in the duodenum. Alpha-1-antytripsine in stool was 6,31 mg/1 g of stool (normal values below 1,81), indicating intestinal protein loss. At that time, the first skin nodules appeared. The patient also underwent gingivectomy, which improved feeding for a short period. Nevertheless after two years, he was dependent on a nasogastric tube for feeding. Despite normal intellectual development, the patient suffered from malnutrition (weight 9,29 kg (-3,25 SD), small height 83,4 cm (-3,12 SD), smaller head circumference 49 cm (-0,94 SD)), nodules on both ears, fleshy nodules and hyperpigmentation in the perianal region, limited joint mobility disabling standing and manipulation, and recurrent diarrhea. At the age of 3 years, joints became less painful with increased mobility, but hypertrophy of soft tissues of the mouth developed, and new skin nodules appeared.

Characterization of the 2F6 monoclonal antibody
To tested whether 2F6 indeed recognizes the CMG2 protein, we silenced CMG2 using a lentiviral vector harboring a cmg2 shRNA in three cell types: human skin fibroblasts, Human Umbilical Vein Endothelial cells (HUVEC) and human Retinal epithelial cells (RPE1). CMG2 could be detected in all three cell lines at the expected ≈50 kDa molecular weight and this band was no longer detected after cmg2 silencing (Suppl. Fig. 1A).
To further characterize the antibody, CMG2 harboring a C-terminal V5 tag was ectopically expressed in Hela cells. Total cells extract whether analyzed by Western blotting under reducing and non-reducing conditions using either an anti-V5 antibody or 2F6. As apparent from Suppl. Fig. 1B, whereas the detection of the V5 tag did not vary when reducing gels were used, detection of WT CMG2 with 2F6 was drastically decreased under reducing conditions. We also analyzed the V310F mutant. This mutation as described in the later part of the main text leads to aberrant disulfide bond formation in the CMG2 Ig-like domain. As for the WT protein, this mutant was detected beter with the 2F6 antibody under non reducing conditions. What is also apparent from Suppl. Fig. 1B, is that the V310F is not detected as efficiently as the WT protein, suggesting that 2F6 is a conformational antibody that preferentially recognize CMG2 with correctly formed disulfide bonds, i.e. properly folded protein.
That 2F6 poorly recognizes the V310F mutant was further confirmed by immunofluorescence. While the V310F mutant could be readily detected with the anti-V5 antibody, no staining was observed with 2F6.