spz (Spätzle, P48607) — review notes

Status of inputs

• UniProt, GOA (91 rows, 89 seeded annotations after de-duplication) and all 35 GOA PMIDs are cached.
• The review was drafted from UniProt, GOA and the cached primary literature; Falcon deep research
  (spz-deep-research-falcon.md) completed afterwards and was checked for consistency.

Identity and structure

• Secreted cystine-knot protein, synthesised as a disulfide-linked dimeric pro-protein; many
  maternal splice isoforms share the C-terminal cystine-knot domain (C-106) and differ in the
  pro-domain [UniProt P48607; PMID:18790733 "Through alternative splicing, the Spätzle gene encodes
  for several isoforms that (with one exception, the "propeptide isoform") share C106 but differ in
  the prosequence."].
• Easter cleaves pro-Spz after Arg220 to release C-106, a disulfide-linked NGF-like dimer
  PMID:9533958.
• During immunity the cleaving enzyme is SPE PMID:16399077; an
  SPE-independent protease can also process Spz PMID:26843333.
• The pro-domain is needed for folding/secretion and masks the Toll-binding determinants
  PMID:17324925.

Molecular function: Toll ligand (cytokine)

• Direct high-affinity binding of mature Spz to the Toll ectodomain; pro-Spz inactive
  PMID:12872120.
• Crystal structures: Spz C-106 dimer binds the concave face of the membrane-distal LRR domain of
  Toll, asymmetric, neurotrophin-like [PMID:24733933 "Spätzle binds to the concave surface of the
  membrane-distal LRR domain"; PMID:24282309 "Spz C106 interacts via its cystine-knot structure in
  an asymmetric binding mode that is reminiscent of the neurotrophin NGF bound to the receptor p75
  NTR"].
• Spz-1 also binds Toll-7 in co-IP and activates the drosomycin promoter through Toll-1 and Toll-7
  in S2 cells PMID:31088910.
• Toll is not a PRR: Spz is an endogenous ligand [PMID:12872120 "These results show that, in
  contrast to the human Toll-like receptors, Drosophila Toll requires only an endogenous protein
  ligand for activation and signaling."; PMID:31088910 "However, vertebrate TLRs do not bind
  cytokines like Spz-1 but instead function as pattern recognition receptors (PRRs)"].

Processes

• Embryonic dorsal-ventral patterning: ventrally processed Spz defines where Toll is active
  [PMID:8124709; PMID:8026333].
• Immunity: Spz/Toll/Cactus control drosomycin and antifungal defence PMID:8808632; required for
  resistance to Gram-positive bacteria PMID:11823479; for defence against fungi and Gram-positive
  bacteria but not Gram-negative bacteria in the DIAP2 study PMID:16894030.
• Haemocyte-derived Spz drives Toll-dependent drosomycin in larval fat body PMID:19934223.
• Neurotrophic roles (with DNT1/DNT2): CNS neuron survival, SNa motor axon targeting
  PMID:19018662.
• Other contexts, all via Toll signalling: epidermal requirement for muscle patterning
  PMID:9676200, cell competition PMID:30146479, tumour response in fat body PMID:24582964,
  wound-induced epidermal gene expression downstream of H2O2 PMID:28289197.

Substrate / participation analysis (proteolysis terms)

• GO:0160032 "Toll receptor ligand protein activation cascade" (part_of GO:0140448 signaling
  receptor ligand precursor processing) describes the protease cascade that cleaves pro-Spz.
• Comparator check (QuickGO, D. melanogaster, 2026-09-30): the term is carried by the proteases
  and recognition factors (SPE, ea, snk, gd, psh, modSP, grass, Hayan, MP1, ndl, pip, GNBP1/3,
  PGRP-SA ...) and not by spz. GO:0140448 likewise sits on the processing enzymes
  (BACE1, ADAM10, ADAM17, MEP1A, CASP1) and not on their substrates.
• Spz supplies no catalytic, scaffold or cofactor work to its own cleavage: it is the substrate
  and product. No NEW annotation to GO:0160032 / GO:0140448 / proteolysis is proposed. The
  enzyme–substrate relationship belongs on SPE/Easter as has input.
• In contrast, the Toll signalling pathway starts with ligand binding to Toll (GO:0008063
  definition), so Spz as the ligand does participate and those rows are accepted.
• No GO-CAM in gocams/index.tsv contains spz.

Questionable rows

• GO:0007310 oocyte dorsal/ventral axis specification (IEP, PMID:8124709): oocyte axis polarity is
  set in the follicle/oocyte upstream (gurken, pipe); spz acts post-fertilisation in the
  perivitelline space. The general GO:0009950 is already present with IDA/IGI support.
• GO:0050829 defense response to Gram-negative bacterium (IMP, PMID:26843333): abstract-only; the
  abstract mentions only M. luteus and B. subtilis (Gram-positive) challenge; conflicting with
  PMID:16894030. Left UNDECIDED.
• GO:0045087 innate immune response (IDA, PMID:17018283): abstract is about human RalB/TBK1; full
  text not cached. Function is clearly correct for spz, so accepted in deference to the curator.
• GO:0010628 positive regulation of gene expression (IMP, PMID:34432851): too general; the paper is
  about the Toll-regulated antifungal Baramicin; proposed MODIFY to GO:0002804.

Deep-research cross-check (2026-09-30)

Compared spz-deep-research-falcon.md with the review.
- Agrees: secreted non-enzymatic cystine-knot cytokine; pro-domain masks receptor binding; Easter (embryo) and SPE/MP1/Hayan/Psh (immunity, damage) processing; C-106 binds the concave LRR face of Toll; Toll-1 is the established receptor and Toll-7 binding is less firmly established; D/V patterning, antifungal and Gram-positive defence; damage-induced activation (consistent with the wounding/H2O2 rows kept as non-core).
- Conflict, resolved in favour of the review: the report calls Spz-Toll binding "low affinity". Primary data say otherwise: "the mature form of Spätzle bound to the Toll ectodomain with high affinity" PMID:12872120 and "an affinity of about 50 nM" for Spz with Toll N13-VLR PMID:24282309. The description's "nanomolar affinity" stands.
- Unresolved: the report's claim that Gram-negative bacteria can activate the pathway draws largely on other insects, so it does not resolve the GO:0050829 row (stays UNDECIDED).
- Paralog statements (Spz2/Spz5 binding Toll-6/7) concern other genes and were not transferred.
- Changes: none to the review file.