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Erythrocyte ? spectrin can be genetically targeted to protect mice from malaria.

ABSTRACT: The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 N-ethyl-N-nitrosourea-induced mouse lines, SptbMRI26194 and SptbMRI53426 , containing single-point mutations in the erythrocyte membrane skeleton gene, ? spectrin (Sptb), which exhibit microcytosis but retain a relatively normal ratio of erythrocyte surface area to volume and are highly resistant to rodent malaria. We propose the major factor responsible for malaria protection is the specific clearance of mutant erythrocytes, although an enhanced clearance of uninfected mutant erythrocytes was also observed (ie, the bystander effect). Using an in vivo erythrocyte tracking assay, we established that this phenomenon occurs irrespective of host environment, precluding the involvement of nonerythrocytic cells in the resistance mechanism. Furthermore, we recapitulated this phenotype by disrupting the interaction between ankyrin-1 and ? spectrin in vivo using CRISPR/Cas9 genome editing technology, thereby genetically validating a potential antimalarial target. This study sheds new light on the role of ? spectrin during Plasmodium infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal adverse consequences for the host.

SUBMITTER: Lelliott PM

PROVIDER: S-EPMC5728631 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Erythrocyte β spectrin can be genetically targeted to protect mice from malaria.

Lelliott Patrick M PM Huang Hong Ming HM Dixon Matthew W MW Namvar Arman A Blanch Adam J AJ Rajagopal Vijay V Tilley Leann L Coban Cevayir C McMorran Brendan J BJ Foote Simon J SJ Burgio Gaetan G

Blood advances 20171212 26

The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 N-ethyl-N-nitrosourea-induced mouse lines, SptbMRI26194 and SptbMRI53426 , containing single-point mutations in the eryth ...[more]

PMID: 29296915

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Project description:Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in ?2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBC(VL)). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic ?- and ?-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrin(VL)) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to ?- and ?-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrin(N)) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrin(N). Although the presence of both N- and O-glycosylations was found both in spectrin(N) and spectrin(VL), enhanced sialylation was predominantly induced in spectrin(VL). Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of ?- and ?-spectrin(VL) confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrin(VL) showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrin(N) suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBC(VL). The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrin(VL) as evidenced by the presence of an additional 60 kDa fragment, absent in spectrin(N) which possibly affects the biology of RBC(VL) linked to both severe distortion of erythrocyte development and impairment of erythrocyte membrane integrity and may provide an explanation for their sensitivity to hemolysis and anemia in VL patients.

Project description:BackgroundDuring erythrocytic schizogony, Plasmodium falciparum interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the P. falciparum M18 aspartyl aminopeptidase (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.MethodsrPfM18AAP was produced as a hexahistidine-fusion protein in Escherichia coli and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-PfM18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.ResultsrPfM18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.ConclusionStudies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

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Erythrocyte ? spectrin can be genetically targeted to protect mice from malaria.

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Erythrocyte β spectrin can be genetically targeted to protect mice from malaria.

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