Galactosidase

GLA activities were determined in Fabry mouse tissues using previously described methods (Desnick et al., 1973 (link)). In brief, tissue samples were homogenized in chilled reporter lysis buffer (Promega) and protease inhibitor (Pierce) was added to the lysates. Protein concentrations were determined using the Bio-Rad Colorimetric Protein Assay Kit. 10 μL of tissue lysate was added to an equal volume of 10 mM 4-methylumbelliferyl-α-D-galactopyranoside (Sigma-Aldrich), dissolved in assay buffer (0.2 M citrate, 0.4 M phosphate buffer, pH 4.4), and 0.1 M N-acetylgalactosamine (Sigma Aldrich), the latter to inhibit α-galactosidase B activity (Mayes et al., 1981 (link)). Following a 30 min incubation at 37°C, reactions were terminated by the addition of 480 μL of 0.1 M ethylenediamine, pH 10.3. The amount of 4-methylumbelliferone (4-MU) produced was determined by measuring fluorescence using a Synergy H1 fluorometer (BioTek). Tissue α-Gal A activities were expressed as nmol of 4-MU produced per h per mg of total protein (nmol/h/mg). Measurement of plasma GLA activities in wildtype mice for PK studies was performed as described above with the following modifications: lysates were incubated with 5 mM 4-methylumbelliferyl α-D-galactopyranoside in assay buffer [20 mM citrate, 30 mM sodium phosphate (pH 4.4), 0.1 M N-acetylgalactosamine, and 4 mg/mL BSA], and the reaction was stopped by addition of stop buffer (0.1 M Glycine, 0.1 N NaOH], as previously described (Shen et al., 2016 (link)).
AGA activity was measured with 1 mM L-aspartic acid β-(7- amido-4-methylcoumarin) in 10% SuperBlock and 90% 50 mM Tris-HC (pH 7.5) for 60 min at 37°C, and then adding 100 µL of stop buffer [0.2 M glycine, 0.175 M NaOH (pH 10.6)], as previously described (Mononen et al., 1993 (link)). GUSB enzyme assay was performed using 10 mM 4-methylumbelliferyl-β-D-glucuronide (Merck) in 0.1 M sodium acetate (pH 4.6) at 37°C for 30 min, and reactions were stopped by 0.1 M sodium carbonate (Grubb et al., 2008 (link)). GAA activity assay was performed with 3 mM 4-methylumbelliferyl-a-D-glucopyranoside (Merck) in assay buffer (30 mM sodium citrate, 40 mM sodium phosphate dibasic, pH 4.0) at 37°C for 3 h (Flanagan et al., 2009 (link)). Reactions were stopped by the addition of an equal volume of 0.4 M glycine, pH 10.8. IDS activity assay was performed with 2.5 mM 4-Methylumbelliferyl sulfate potassium salt (Merck) in 50 mM sodium acetate, at 37°C for 4 h (Dean et al., 2006 (link)). Reactions were stopped with glycine carbonate buffer (pH 10.7). Fluorescence was measured by microplate reader with 360/40 nm excitation and 440/30 nm emission filters.

To determine the site required for the interaction of MaCFEM85 with MsWAK16, PCR amplification was performed to generate multiple truncated forms of MaCFEM85. The CFEM domain (MaCFEM85-CFEM; aa residues 19–86) and the C terminal without the CFEM domain (MaCFEM85-C, aa residues 87–170) were inserted into the vector pGBKT7 as the bait protein (Supplementary Table S1). Another five variants were constructed using polypeptide synthesis to mutate cysteine to alanine at positions 26, 30, 43, 52, and 26/30/43/50/52/64/69/85 (ΔCFEM85₂₆, ΔCFEM85₃₀, ΔCFEM85₄₃, ΔCFEM85₅₂, and ΔCFEM85_{8 all}, respectively). These variants were used to perform Y2H experiments with MsWAK16-ED. The transformed yeast cells were assayed for growth on synthetic dropout SD/-Trp-Leu plates and SD/-Trp-Leu-His-Ade plates containing X-α-Galactosidase (X-α-Gal).