DEV030775 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Antibodies specifically identify their respective isozyme. (A) Alignment of the eTG and nTG open reading frames, indicating identical amino acids residues (dots). Colored outlines show the epitopes used to generate the respective antisera. (B) Schematic of epitope domains that each anti-transglutaminase population recognizes. Colors are as in ‘A’. Bar equals 100 residues. (C) Immunoblots probing catalytically inactive (Antonyak et al., 2006; Mian et al., 1995) full-length versions of each transglutaminase synthesized by in vitro transcription-translation using rabbit reticulocyte lysate from the SP6 TNT Quick Coupled Transcription/Translation System (Promega Corporation, Madison, WI, USA), with sera (left; preimmune serum (1:200 dil (v/v), anti-TG sera eTG, 1:200 dil (v/v) or nTG, 1:500 dil (v/v) or affinity-purified IgGs (right; preimmune, 0.5 µmg/ml, eTG, 0.5 µg/ml or nTG, 0.1 µg/ml) raised against the respective isozymes. The same set of blots was also probed for the myc epitope as a loading control, using monoclonal antibody 9E10 Covance Incorporated, Princeton, NJ, USA; 1:5000 dil (v/v). Specific anti-transglutaminase detection was accomplished using Pierce Super Signal substrates (Thermo-Fisher Scientific Incorporated, Rockford, IL, USA), whereas anti-myc detection was achieved with a luminol-hydrogen peroxide solution 1.25 mM luminol, 68 µM coumeric acid, 0.0093% hydrogen peroxide (v/v), 0.1 M Tris (pH 8). Digitized film images were inverted and then band intensity was quantified with Metamorph software (Molecular Devices, Sunnyvale, CA, USA). Nonspecific signal from the eGFP-myc lanes were subtracted from the TG-myc lanes, and this value was normalized to the total load of protein (anti-myc signal). The TG:myc ratio was then totaled per blot, and the percentage of specific signal shown was calculated from these values. (C′) An extended exposure of the anti-nTG sera blot, indicating the maximal level of antibody cross-reactivity measured (10%) by this method. (D) Schematic of each protein synthesized in vitro for the specificity test (C). Bar equals 100 residues.

• Supplemental Figure S2 -

  Fig. S2. Titration of anti-transglutaminase activity on cadaverine-AlexaFluor488 incorporation into the fertilization envelope. Representative images of fertilization envelopes generated in the presence of 0, 125, 250 or 500 pg of affinity-purified anti-TG per egg. Images show reporter cadaverine-AlexaFluor488 (top) and tyramide-AlexaFluor594 (middle) incorporation into the same fertilization envelope, measuring transamidation and ovoperoxidase-dependent dityrosine crosslinking, respectively. Corresponding DIC images are shown along the bottom row.

• Supplemental Figure S3 -

  Fig. S3. Mass spectrometry peptide matches for cadaverine-AlexaFluor488 spots. Amino acid sequences of the major fertilization envelope protein identified by mass spectrometry from spots containing cadaverine-AlexaFluor488. Peptide matches with >95% confidence are highlighted in bold. Note that a peptide (‘PYEPQDVRPPEPQNVR’) identified by mass spectrometry is not present in the published sequence of S. purpuratus SFE9, but does exist in the coding sequence predicted from the raw genome. This result suggests that the original cDNA clone (Wessel, 1995) may be erroneous as a single frame shift upstream of the ‘NHK’ repeats results in the ‘PYE’ sequence.

• Supplemental Figure S4 -

  Fig. S4. Complete dataset showing the effect of specific enzyme inhibitors on the activity of transamidation, hydrogen peroxide production and dityrosine crosslinking. (A) Schematic listing methods of measuring specific enzyme activities through fluorescent reporters. (A′) List of pharmacological inhibitors used to knock down specific enzymes used during fertilization envelope maturation. Effects of specific inhibitors on hydrogen peroxide production by Udx1 (resorufin conversion by supplemental horseradish peroxidase) using pharmacological inhibitors (B) or affinity purified anti-TG IgGs (B′); on ovoperoxidase activity resorufin conversion by endogenous ovoperoxidase (C) or tyramide-AlexaFluor594 incorporation into individual fertilization envelopes (C′); and on transamidation by transglutaminase (cadaverine-AlexaFluor488 incorporation into individual fertilization envelopes (D). Individual inhibitors are listed along the y-axis, in purple. Antibody inhibition is listed in green or red. All data normalized to respective controls (ASW or preimmune IgGs). Standard deviations (vertical bars) for at least four independent experiments representing 12 pools of 200 eggs (resorufin) or 20 individual fertilization envelopes (AlexaFluor reporters) are shown. Results of Student’s t-test are shown when significantly different from control (P≤0.01).