PRSS55

PRSS55 is a testis-specific chymotrypsin-like (S1 family) serine protease, defined by a catalytic triad of His108, Asp156, and Ser250 and a C-terminal hydrophobic segment that supports membrane anchoring, expressed in testicular spermatids and epididymal spermatozoa (PMID:18844450, PMID:33417308). It is essential for male fertility: Prss55-deficient sperm fail to migrate through the uterotubal junction and cannot bind the zona pellucida, a phenotype that tracks with near-complete loss of mature ADAM3 protein from epididymal sperm despite an unchanged ADAM3 precursor in testis (PMID:30032357, PMID:32301961). PRSS55 does not co-immunoprecipitate with ADAM3, so it stabilizes mature ADAM3 indirectly rather than as a direct binding partner, and genetic epistasis with the related Prss51 establishes that PRSS55, not PRSS51, carries this fertility requirement (PMID:30032357, PMID:32301961). Beyond the ADAM3 axis, loss of PRSS55 compromises sperm structural integrity, producing defective '9+2' axonemal microtubules, damaged peripheral dense fibre, and abnormal mitochondrial cristae alongside reduced motility (PMID:33417308). A second functional role situates PRSS55 in mitochondrial energy metabolism: it localizes to mitochondria, its loss lowers ATP production and mitochondrial membrane potential while its overexpression raises ATP and the NAD+/NADH ratio, and it physically interacts with the branched-chain amino acid metabolic enzymes BCKDK and BCKDHA, with Prss55-/- testes accumulating valine, leucine, and isoleucine (PMID:41444608). In humans, a homozygous missense mutation (p.A192V) destabilizes the protein and causes teratozoospermia with head, neck, midpiece and tail defects, and human PRSS55 functionally rescues the mouse knockout in a manner dependent on C-terminal membrane topology (PMID:35821214, PMID:40764777).

1. 2008 Medium

   Established the molecular identity of PRSS55 as a membrane-anchored S1-family serine protease, defining its catalytic triad and testicular expression and framing it as an enzyme rather than an inert structural protein.

   Evidence RACE cDNA cloning, sequence/domain analysis, and immunohistochemistry with a specific antibody in testis

   PMID:18844450

   Open questions at the time

   • No proteolytic substrate or demonstrated enzymatic activity
   • Membrane-anchoring inferred from a hydrophobic segment, not experimentally resolved
   • No fertility or loss-of-function phenotype tested
2. 2018 High

   Connected PRSS55 to male fertility by showing knockout sperm fail uterotubal migration and oocyte binding, and tied this to indirect loss of mature ADAM3, defining the protein's reproductive function and its non-direct relationship to ADAM3.

   Evidence Prss55-/- mice with in vivo/in vitro fertilization assays, Co-IP, western blot for ADAM3, and microarray

   PMID:30032357

   Open questions at the time

   • Mechanism by which PRSS55 stabilizes mature ADAM3 indirectly is undefined
   • No identified protease substrate linking enzymatic activity to ADAM3 maturation
3. 2018 Low

   A contradictory CRISPR knockout reported normal fertility and histology, raising the question of whether the infertility phenotype was robust or line-dependent.

   Evidence CRISPR/Cas9 knockout with fertility mating trials and histology

   PMID:29563520

   Open questions at the time

   • Minimal phenotypic characterization; contradicted by multiple subsequent independent knockouts
   • Possible incomplete gene disruption not excluded
4. 2020 High

   Resolved the contradiction and refined gene assignment by using genetic epistasis to show PRSS55 single knockouts phenocopy Prss51/Prss55 double knockouts while Prss51 single knockouts are fertile, pinning the ADAM3-stabilizing fertility requirement specifically on PRSS55.

   Evidence CRISPR/Cas9 single and double knockouts with UTJ migration, ZP binding assays, and ADAM3 western blot

   PMID:32301961

   Open questions at the time

   • Biochemical pathway from PRSS55 to mature ADAM3 still unmapped
   • Does not address structural or metabolic roles
5. 2021 Medium

   Broadened the phenotype beyond ADAM3 by demonstrating that PRSS55 loss disrupts sperm ultrastructure (axoneme, dense fibre, mitochondrial cristae) and lowers ATP and electron transport components, implicating PRSS55 in sperm structural integrity and energy metabolism.

   Evidence Whole-transcript Prss55 knockout, TEM ultrastructure, ATP measurement, and western blot for electron transfer chain components

   PMID:33417308

   Open questions at the time

   • Myosin-activation mechanism stated as preliminary
   • Causal link between protease activity and structural/metabolic defects not established
6. 2022 Medium

   Extended the function to humans by identifying a destabilizing missense mutation causing teratozoospermia, confirming PRSS55 protein abundance is required for normal sperm morphology across species.

   Evidence Whole-exome and Sanger sequencing, Papanicolaou staining, SEM/TEM, immunofluorescence, and western blot in an infertile patient

   PMID:35821214

   Open questions at the time

   • Single patient; causality rests on protein destabilization and mouse parallels
   • No biochemical test of mutant enzymatic or anchoring function
7. 2025 Medium

   Tested functional conservation and topology requirements by transgenic rescue, showing human PRSS55 replaces mouse PRSS55 and that the C-terminal tag's membrane orientation governs rescue efficiency.

   Evidence Transgenic rescue in Prss55 knockout mice with intracellular-tagged versus GPI-anchored extracellular-tagged constructs and mating trials

   PMID:40764777

   Open questions at the time

   • Partial rescue with the GPI construct not mechanistically explained
   • Native topology of endogenous PRSS55 not definitively settled
8. 2025 Medium

   Provided a second mechanistic axis by localizing PRSS55 to mitochondria and showing it physically interacts with the BCAA metabolic enzymes BCKDK and BCKDHA, linking PRSS55 to oxidative phosphorylation and ATP homeostasis in sperm.

   Evidence Immunofluorescence and fractionation, overexpression with ATP/NAD+ assays, proteomics, metabolomics, LC-MS/MS, and Co-IP

   PMID:41444608

   Open questions at the time

   • Mitochondrial localization is in tension with earlier GPI-anchored surface localization claims
   • Whether PRSS55 proteolytically processes BCKDK/BCKDHA or binds non-catalytically is unknown
   • Single-study finding without independent replication

• The unifying biochemical activity of PRSS55 remains unknown: no direct proteolytic substrate has been identified, and it is unresolved how a single protein reconciles GPI-anchored surface localization, mitochondrial residence, indirect ADAM3 stabilization, and BCAA-enzyme binding.
• No demonstrated protease substrate
• Conflicting subcellular localization reports unreconciled
• Mechanistic link between protease identity and metabolic/structural phenotypes absent