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H05 — DAF SCR1-4 CP0-Closure Thesis (Stub)

Stub status. Committed 2026-05-05 to register the hypothesis in the falsification-card directory and force the "what would kill this thesis" framing onto the just-closed (computationally) CP0 gap. Full population (assumption stack, killshot menu, pre-committed thresholds, kill switches, failure-mode coverage map) queued as a Phase 2 item — see complement-c5a-gout.md § CP0 status update and daf-cd55-scr14-truncated-computational.md.

Pre-registration discipline (per H01) does not apply until this stub is upgraded to a full card.

Coordination with comp-018 (2026-05-08): The upstream complement modulator sweep operates at the same chokepoint (CP0) but a different angle (compound-class breadth at upstream nodes vs. engineered-DAF at C3 convertase decay-acceleration). H05's thesis is NOT undermined by comp-018; the two threads are mechanistically complementary. Specifically: dietary rosmarinic acid (TIER-1 C3 convertase inhibitor, IC50 5-10 µM) and engineered DAF SCR1-4 act at different geometric scales — RMA in fluid phase + gut lumen, DAF on the MSU surface. comp-018 also surfaces C1-INH as a near-twin engineering thesis (different soluble human complement regulator, different node, FDA-approved precedent), which becomes H05's sister-hypothesis card if C1-INH protease stability passes a future comp-NNN computational gate.

Claim (provisional, stub-level)

A soluble DAF/CD55 SCR1-4 truncated construct (UniProt P08174, residues 35–285) heterologously expressed in A. oryzae via the standard Ward 1995 / Huynh 2020 secretory cassette architecture, delivered in shio-koji or dried-koji format, is a viable fermentable CP0 (complement priming) modulator that closes the Open Enzyme platform's documented complement-coverage gap.

The thesis composes three sub-claims, each independently falsifiable:

  1. Cassette feasibility. The SCR1-4 construct can be cloned with a koji-native α-amylase signal peptide, expressed in A. oryzae (RIB40 or NSlD-ΔP10 protease-deletion host) at therapeutic-relevant titers (≥50 mg/L pore-fluid equivalent, mirroring the H01 lactoferrin floor scaled for the smaller protein), and secreted as a correctly-folded soluble fragment with all 8 intrachain disulfide bonds (2 per SCR domain × 4 SCRs, per UniProt P08174) intact.

  2. Functional CCP-regulatory activity. The expressed soluble truncated fragment retains decay-accelerating function — specifically C3b and C4b binding, and C3 convertase decay-acceleration sufficient to suppress C5a generation in a complement-activation assay (e.g., zymosan-stimulated human serum + ELISA for C5a).

  3. Mucosal-surface delivery. Luminal-side soluble DAF SCR1-4 (delivered orally as part of an engineered koji product) actually engages the gout-relevant complement-priming step in the gut → submucosal macrophage signaling axis, OR alternatively acts at the bacterial/mucus complement-priming interface in a manner that meaningfully reduces downstream CP0 priming load. The macrophages doing the CP0 priming are submucosal; whether luminal DAF can reach them or only modulate proximal complement activation is empirically open.

The truncation is grounded in comp-012: the full DAF ectodomain (aa 35–353) failed comp-006 protease stability (HIGH risk, max 0.388 — driven by the disordered Ser/Thr stalk aa 286–353); the truncated SCR1-4 construct (aa 35–285) tested LOW (0.039, identical to uricase) with 100% of exposed sites eliminated. Computational feasibility is in silico-validated; the three sub-claims above are the wet-lab gating questions.

Assumption Stack (placeholder — to be populated when this stub is upgraded)

Anticipated load-bearing assumptions:

  1. Disulfide folding fidelity. A. oryzae ER PDI capacity is sufficient to fold 8 intrachain disulfides on a single ~28 kDa secreted protein. Comp-010 noted the OE pair (uricase 0 disulfides + lactoferrin 17 disulfides = 17 total) is within demonstrated NSlD-ΔP10 capacity per Huynh 2020. Adding DAF SCR1-4's 8 disulfides (per UniProt P08174, corrected from an earlier hallucinated estimate of 12; see comp-012 §1.5 correction note) as a third co-expressed cassette would push to 25 total — a real but not catastrophic increase, and ~14% lower than the prior estimate.

Triple-cassette synergy prediction (revised 2026-05-06 — per-domain architecture refinement): The chaperone-orthogonal stacking framework §5.5 makes a prospective falsifiable prediction for the triple-cassette (uricase + Lf + DAF SCR1-4) scenario. The initial bulk-count prediction (0.45–0.70, central 0.55–0.65) has been revised downward by the §3.5 per-architecture PDI residence time refinement. Revised prediction: 0.35–0.65 (central expectation 0.45–0.55), with Lf's transferrin-lobe slow-folding architecture as the dominant bottleneck (not raw disulfide count). Key insight from the architecture refinement:

  • Lf's effective PDI load = 16 disulfides × α(1.5–2.5) = 24–40 (architecture-adjusted), substantially above its bulk count of 16 implies
  • DAF SCR1-4's effective PDI load = 8 disulfides × α(0.3–0.6) = 2.4–4.8, substantially below its bulk count implies (compact CCP modules fold quickly)
  • Combined triple-cassette effective PDI load = 26.4–44.8 vs. Huynh reference 16.0 (= 1.65–2.80× Huynh), compared to prior bulk estimate of 25/16 = 1.56×
  • The architecture refinement increases the effective load range (from 1.56× fixed to 1.65–2.80×), driving the revised central prediction below the prior range

The three decision gates (framework-convention, not empirically derived):

  • Synergy >0.85 (very low probability, lower than bulk model): pursue triple-cassette endgame strain as the single-strain CP0+CP1-CP6 solution. Requires both the Huynh ceiling to be strongly antibody-architecture-specific AND Lf's in vivo ER-assisted folding to be substantially faster than its in vitro transferrin-lobe kinetics imply (i.e., α ≈ 1.0 in a functional secretory pathway).
  • Synergy 0.6–0.85 (low–medium probability): pursue triple-cassette with PDI co-expression helper augmentation (4-cassette design). Achievable only if Lf's α is at the favorable end (1.5) AND §1.9 Lf-alone arm confirms >500 mg/L (resolving the Huynh ambiguity favorably). Single PDI overexpression captures ~1.05–1.15× rescue (intra-paper Zhang 2006 PMID 16889384); combination helpers add 1.2–1.5× over singles.
  • Synergy <0.6 (high probability, higher than bulk model): DAF cassette goes on a separate strain (two-strain co-fermentation) or onto the engineered LBP chassis as a parallel peer track. H05 stays alive in this case — only the chassis-route changes. The architecture refinement shifts the central expectation firmly into this gate. The endgame strain remains uricase + Lf (CP1-CP6 coverage); CP0 direct-antagonism coverage becomes a separate-strain or peer-track output.

The falsifiable test: measure Lf titer in the triple-cassette strain vs. the dual-cassette (uricase + Lf) baseline in the same §1.9 wet-lab experiment. If Lf titer in the triple is >85% of the dual-cassette baseline, the triple stacks cleanly. If <60%, separate-strain routing is recommended. The §1.9 Lf-alone arm must run first to resolve the capacity-vs-titer benchmark ambiguity (§8 item 7) before interpreting any dual or triple result — if Lf alone reaches >500 mg/L in NSlD-ΔP10 solid-state, the upper bound of the triple prediction shifts more favorably. See chaperone-orthogonal-stacking.md §5.5 for the full bounded analysis. 2. CCP-regulatory function survives truncation. Native DAF/CD55's decay-accelerating function uses all four SCR domains plus the membrane GPI anchor for proper geometry; truncating to soluble SCR1-4 changes the geometry. Some published soluble DAF constructs retain function; whether the specific aa 35–285 boundary preserves activity is not pre-validated. 3. Mucosal access geometry. Submucosal macrophages doing CP0 priming are on the basolateral side of the epithelium; luminal-side soluble DAF would need to either cross the epithelium (unlikely for a 28 kDa protein) or modulate complement upstream (in the lumen, on bacterial surfaces, in the mucus layer) in a way that meaningfully reduces priming load reaching the macrophages. 4. Alternative-pathway dominance in gout-relevant complement priming. DAF inhibits both classical and alternative pathway C3 convertases; if the gout-relevant priming is driven primarily by a pathway DAF doesn't cover (e.g., MBL-pathway dominance or direct C5 cleavage), the inhibition is incomplete. 5. Ferment-stability of the active form. comp-012 verifies protease stability of the polypeptide backbone in shio-koji; whether the disulfide-folded active form survives ALL of fermentation + storage + gut transit + reaching the colonic activity site is empirically open.

Killshot Menu (placeholder — to be populated when this stub is upgraded)

Anticipated highest-priority killshots:

  • Wet-lab expression in A. oryzae RIB40 first (~$2K, ~6 weeks): is the construct secreted at all? SDS-PAGE under non-reducing conditions for disulfide assessment.
  • CCP-regulatory activity assay on the secreted fragment (~$1K reagents + assay): does the truncated soluble form retain function?
  • Literature deep-dive on published soluble DAF constructs ($0, ~1 week — could be a Paperclip-grep follow-up per etc/manual-literature-mining.md): has anyone made and tested an aa 35–285 soluble DAF? What was the activity profile?
  • Comparison with sCR1 / Factor H truncated soluble constructs ($0): if other complement regulators have published soluble-truncated activity precedents, that informs the DAF design space.

Pre-Committed Thresholds (placeholder — to be populated when this stub is upgraded)

Anticipated structure: - Alive: secreted ≥50 mg/L pore-fluid equivalent + ≥40% native disulfide-folded form on non-reducing SDS-PAGE + ≥30% C5a-generation inhibition vs. control in a zymosan-activation assay - Killed: secreted <10 mg/L OR fully reduced (no disulfides) OR no detectable CCP-regulatory activity - Pending: intermediate values; iterate on construct boundaries (try aa 35–250 alternative truncation), host strain (NSlD-ΔP10 vs. RIB40), or secretion signal

Status

Stub. Computational claim verified by comp-012 (LOW protease risk, 2026-05-05). Wet-lab not yet executed. Falsification card to be upgraded to full when wet-lab access is confirmed and a sub-experiment is committed.

Survival count: 0.

Survival score: 0.0 (undefined until full card and first survived killshot).

Cross-References