A0A2G9RZF1 bioinformatics: identity of the 156-aa CUB ORF (re-review, 2026-10-21)

Scripts (reproducible; need curl and HMMER3 phmmer):

fetch_and_phmmer.sh fetches the query and a panel of 17 CUB proteins from UniProt
and runs phmmer (output phmmer_panel.out, phmmer_panel_summary.tsv).
refseq_compare.sh fetches the complete bullfrog TLL1 (XP_073462190.1, 1005 aa)
and BMP1 (XP_073478370.1, 1020 aa) RefSeq models from the chromosome-level
annotation and runs phmmer (output phmmer_refseq.out, phmmer_refseq_summary.tsv).
cub5_identity.py counts identical aligned positions in every phmmer domain
alignment above 100 bits (cub5_identity.tsv, cub5_identity_refseq.tsv).

Result 1: the ORF is the C-terminal CUB5 of a tolloid

In every full-length tolloid in the panel the query's best domain hit is the
last CUB domain (CUB5), and the alignment runs to the protein's C-terminus. For
human TLL1 (1013 aa) query 30-156 aligns to 886-1012 at 229.3 bits. Hits to
CUB1-CUB4 of the same proteins and to PCOLCE1/2, MFRP, NETO1/2 and cubilin CUB
domains all score 80 bits or less.

Target (UniProt)	PANTHER subfamily	bits	identity
Q8JI28 TLL1_XENLA	SF45	260.4	110/127 (86.6%)
O43897 TLL1_HUMAN	SF45	229.3	99/127 (78.0%)
Q62381 TLL1_MOUSE	SF45	217.0	94/127 (74.0%)
P13497 BMP1_HUMAN	SF53	208.5	86/126 (68.3%)
P98063 BMP1_MOUSE	SF53	207.9	86/126 (68.3%)
O57382 TLL2_XENLA	SF45	203.7	85/126 (67.5%)
Q9Y6L7 TLL2_HUMAN	SF45	196.9	85/128 (66.4%)

Identity tracks the species tree within the TLL1 orthogroup (frog > mammal TLL1)
and is lower for every BMP1 and TLL2 paralog. That is the pattern of a TLL1
ortholog, not just a best hit.

Result 2: it matches the complete bullfrog TLL1 gene model almost exactly

RefSeq model	bits (CUB5)	identity	target span
XP_073462190.1 tolloid-like protein 1 (1005 aa)	287.7	126/127 (99.2%)	879-1005
XP_073478370.1 bone morphogenetic protein 1 (1020 aa)	164.1	71/139 (51.1%)	893-1018

A0A2G9RZF1 residues 30-156 are therefore the last 127 residues (CUB5) of
bullfrog TLL1. Residues 1-29 do not align to either model. They are probably an
intronic or untranslated read-through from the fragmented draft gene model.

Interpretation

A0A2G9RZF1 is a fragmentary gene model of bullfrog TLL1. It is 156 aa against
1005 aa for the complete product (about 13% of the protein). It lacks the
signal peptide, the prodomain, the astacin-like zinc metalloprotease domain
(and with it every catalytic residue), CUB1-CUB4 and both EGF-like domains.
UniProt's PANTHER placement (PTHR24251:SF37 "CUB DOMAIN-CONTAINING PROTEIN")
is a fragment artefact. The tolloid subfamily HMMs (SF45 holds TLL1 and TLL2;
SF53 holds BMP1) model the full multi-domain protein, so a lone CUB domain
falls into a heterogeneous single-CUB bin. Note that UniProt places the
neighbouring fragments on scaffold KV928989 in SF45 (A0A2G9RZH1) and SF53
(A0A2G9RZI6), which is no more reliable for subfamily assignment.
The fragment has no catalytic activity of its own. Any function annotation
belongs to the TLL1 gene.