Bioinformatics Analysis Identifies Sequence Determinants of Enzymatic Activity for the PHARC-Associated Lipase ABHD12

In humans, PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract)
is an early onset autosomal recessive neurological disorder caused
by deleterious mutations to ABHD12 (α/β-hydrolase domain
protein # 12). Biochemically, ABHD12 functions as a lipase and catalyzes
the hydrolysis of lysophosphatidylserine (lyso-PS) (lyso-PS lipase).
By doing so, it controls the concentrations and signaling pathways
regulated by this potent signaling lysophospholipid in the mammalian
brain. While genetic mapping efforts have identified over 30 mutations
in ABHD12 from human PHARC subjects, the biochemical activity of these
pathogenic mutants remains unknown. To understand this, here, we performed
an exhaustive bioinformatics survey and collated ABHD12 protein sequences
from various organisms across evolution. Next, based on sequence alignments
and structural modeling, we identified functionally relevant conserved
residues in the ABHD12 protein sequence that are potentially important
for its enzymatic activity. To validate these in silico findings,
we generated numerous mutants of murine ABHD12, including those associated
with human PHARC subjects, and assayed them for their enzymatic activity.
Taken together, these complementary in silico and biochemical studies
provide the first thorough sequence-function relationship for mammalian
ABHD12, especially relevant in the context of PHARC. Finally, our
evolutionary analysis identified CG15111 as an ABHD12 ortholog in
the fruit fly (Drosophila melanogaster), and enzymatic assays indeed confirmed that recombinant CG15111
has robust lyso-PS lipase activity. Flies serve as an excellent animal
system to model various human neurological diseases, and the identification
of CG15111 as a Drosophila melanogaster ABHD12 ortholog opens new avenues to study PHARC in fly models.