Annotation inferences using phylogenetic trees

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

PMID:36241929

Malaria parasites utilize two essential plasma membrane fusogens for gamete fertilization.

Plasmodium genomes encode two HAP2/GCS1 family proteins; this gene, PF3D7_1014200, is PfHAP2, while PF3D7_0816300 is the paralog PfHAP2p. Evidence must be assigned to the correct paralog.

"This revealed two distinct HAP2/GCS1-encoding genes in all Plasmodium species, PfHAP2 (PlasmoDB ID PF3D7_1014200) and PfHAP2p (paralog of HAP2, PlasmoDB ID PF3D7_0816300)"
GFP-tagged PfHAP2 is expressed internally in male stage V gametocytes and is displayed throughout the flagellum of the microgamete, and is not detected in female gametocytes.

"This revealed that PfHAP2 was expressed internally in male stage V gametocytes while in the microgametes, it was expressed throughout the flagellum (Fig. 2A)."
PfHAP2 expression is male-restricted at the gametocyte stage.

"PfHAP2 expression was not detected in female stage V gametocytes (Fig. 2B)."
CRISPR/Cas9 deletion of PfHAP2 leaves asexual growth, gametocytogenesis and exflagellation intact but abolishes zygote formation.

"These results indicate that both PfHAP2 and PfHAP2p are individually essential for zygote formation."
Microgamete formation is unaffected in the knockout, localizing the defect to fertilization rather than to male gamete development.

"There were no significant differences observed in the number of exflagellation centers of Pfhap2¯ and Pfhap2p¯ and WT PfNF54 control (Fig. 3C)."
Pfhap2-null parasites produce no mosquito midgut oocysts, so the gene is required for transmission to the vector.

"showed no oocysts in the Pfhap2¯ and Pfhap2p¯ lines, demonstrating that they are each required for parasite transmission to mosquitoes (Fig. 4A)."
Genetic crosses with male-only and female-only producer lines show that the requirement for PfHAP2 is male-restricted.

"These results show that Pfhap2¯ males are sterile but females are fertile."
Heterologous expression of full-length PfHAP2 in BHK cells drives cytoplasmic content mixing and multinucleation, demonstrating intrinsic fusogenic activity comparable to Arabidopsis HAP2.

"We found that both PfHAP2 and PfHAP2p induced cytoplasmic content mixing (i.e., cell fusion) to similar levels as the AtHAP2 (Fig. 5A–C)."
In the heterologous assay fusion requires the fusogen on both membranes, whereas the genetic crosses indicate a male-only requirement in the parasite; the directionality of HAP2-driven gamete fusion in vivo is therefore unresolved.

"The proteins must be present in both fusing cells."

PMID:18367645

The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes.

HAP2 acts specifically at the bilayer merger step: membrane dye transfer is blocked in hap2 mutants although the gametes attach normally.

"Membrane dye experiments show that HAP2 is essential for membrane merger."
HAP2 is dispensable for species-specific recognition and for the tight prefusion attachment that precedes fusion, in both Chlamydomonas and Plasmodium.

"hap2 gametes in both Chlamydomonas and Plasmodium recognized and adhered to gametes of the opposite sex indicated that HAP2 is not essential for species-limited membrane recognition or the tight, prefusion attachment of the fusogenic membranes that precedes membrane fusion."

PMID:18403203

Male fertility of malaria parasites is determined by GCS1, a plant-type reproduction factor.

Knockout of the P. berghei HAP2/GCS1 ortholog produces sterile males and failed fertilization, establishing the male-restricted requirement later confirmed in P. falciparum.

"PbGCS1 knockout parasites show male sterility, resulting in unsuccessful fertilization."

PMID:28235200

The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

Crystallography of Chlamydomonas HAP2 shows the family adopts a class II viral fusion protein fold, and blocking the fusion loop abolishes gamete fusion, establishing HAP2 as the fusogen itself rather than an accessory factor.

"targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion."

PMID:29212032

Targeting the Conserved Fusion Loop of HAP2 Inhibits the Transmission of Plasmodium berghei and falciparum.

Antibodies raised against the HAP2 cd (fusion) loop stain the surface of non-permeabilized activated male gametes and the staining is lost in HAP2 knockout parasites, showing that HAP2 is displayed on the microgamete plasma membrane in its native fold.

"Staining was absent in assays with HAP2-KO parasite lines, indicating the ability of these antibodies to recognize natively folded HAP2 on the gamete surface (Figures 2A and 2B)."
Antibodies against the P. falciparum HAP2 cd loop reduce P. falciparum transmission in field membrane-feeding experiments.

"A corresponding reduction in P. falciparum transmission (75.5%/36.4% reductions in intensity/prevalence) is observed in complimentary field studies."

PMID:41498187

Crystal structure and nanobodies against domain 3 of the malaria parasite fusogen Plasmodium falciparum HAP2.

Nanobodies binding domain 3 of PfHAP2 were isolated and the crystal structure of one complex was solved, giving the first experimental structure of any PfHAP2 domain (PDB 9YC3).

"We have identified nanobodies that bind specifically to domain 3 of PfHAP2 with nanomolar affinities, two of which show some cross-species reactivity with HAP2 of other Plasmodium species. The crystal structure of one nanobody in complex with domain 3 of PfHAP2 provides the first structural insights into this transmission-blocking target in P. falciparum."
The field consensus, restated by these authors, is that PfHAP2 is essential for gamete fusion during fertilization and is a transmission-blocking target.

"PfHAP2 is essential for malaria parasite gamete fusion during fertilisation, but its potential as a transmission-blocking candidate has not been fully explored [2]."

PMID:24042109

Functional comparison of Plasmodium falciparum transmission-blocking vaccine candidates by the standard membrane-feeding assay.

IgG raised against recombinant PfHAP2 inhibits oocyst intensity in the standard membrane-feeding assay comparably to anti-Pfs25 and anti-Pfs230, independent functional evidence that PfHAP2 is required at the gamete-to-oocyst transition.

"When IgGs purified from pools of immune serum were tested at 0.75 mg/ml in the SMFA, all three IgGs showed 97 to 100% inhibition in oocyst intensity compared to control IgG."

file:PLAF7/HAP2/HAP2-uniprot.txt

UniProtKB Q8IJQ3 (Q8IJQ3_PLAF7) flat file

UniProt assigns the protein to the HAP2/GCS1 family and annotates a single-pass type I cell-membrane topology, with a predicted transmembrane helix at residues 682-700 and a disordered, basic cytoplasmic region.

"Belongs to the HAP2/GCS1 family."