ANTXR2 Human

What is ANTXR2 and what is its primary function in humans?

ANTXR2 encodes a cell surface protein initially identified as a receptor for Bacillus anthracis virulence toxins, which are necessary for causing anthrax disease. In humans, ANTXR2 has undergone significant evolutionary changes compared to non-human primates, particularly in immune cells where it shows approximately 8-fold lower expression . Beyond its role in anthrax pathogenesis, ANTXR2 has been implicated in extracellular matrix (ECM) homeostasis, particularly in reproductive tissues, through regulation of matrix metalloproteinases (MMPs) .

How does ANTXR2 expression differ between humans and non-human primates?

Comparative genomic analyses have revealed that ANTXR2 is transcribed at significantly lower levels (approximately 8-fold reduction) in human CD4+ T cells compared to non-human primates like chimpanzees, rhesus macaques, and baboons (p<0.0001) . This decreased expression has been confirmed across 91 human samples, suggesting this is a human lineage-specific change rather than individual variation. The downregulation appears to be primarily limited to immune cells, which may reflect adaptation to anthrax exposure during human evolution .

What experimental systems are used to study ANTXR2 function?

Researchers employ several model systems to study ANTXR2 function:

• Cell culture models: Human cell lines (HEK293T, K562) and immune cells (CD4+ T cells) for expression studies and receptor function

• Mouse models: Female mice lacking ANTXR2 have been used to study reproductive functions

• Protein interaction studies: Surface plasmon resonance (SPR) experiments and pull-down assays to study binding properties

• Gene editing: CRISPRi has been used to knock down regulatory elements affecting ANTXR2 expression

What evidence suggests evolutionary selection has acted on ANTXR2 in humans?

Multiple lines of evidence indicate evolutionary selection on ANTXR2 in humans:

• Expression divergence: The consistent 8-fold downregulation in human immune cells compared to multiple non-human primate species suggests a human-specific adaptation .

• Cis-regulatory divergence: Multiple cis-regulatory elements (CREs) around the ANTXR2 locus show decreased transcription in humans compared to non-human primates, with a median 2.3-fold lower transcription (p=0.001) .

• Recent positive selection: Genetic signatures consistent with recent positive selection have been identified, particularly a European-specific decrease in ANTXR2 expression across multiple tissues affected by anthrax toxins .

How might changes in ANTXR2 reflect human adaptation to disease exposure?

The evolutionary changes in ANTXR2 expression appear to follow two distinct periods of adaptation:

• Early human evolution: The consistent downregulation in immune cells across all human samples suggests an early adaptation, possibly related to increased pathogen exposure associated with hunting and scavenging behaviors in early humans .

• Agricultural transition: Evidence of more recent positive selection, particularly in European populations, suggests a second period of adaptation following the rise of modern agriculture, which may have increased anthrax exposure through closer contact with domesticated animals .

As ANTXR2 is necessary for anthrax toxin entry into cells, reduced expression may confer resistance to anthrax infection, representing an evolutionary adaptation to changing disease exposure patterns in human history .

What role does ANTXR2 play in mammalian reproduction?

Research using mouse models has demonstrated that ANTXR2 is necessary for successful pregnancy and labor:

• Female mice lacking ANTXR2 could become pregnant but were unable to deliver, indicating a critical role in the labor process .

• ANTXR2 regulates matrix metalloproteinases (MMPs) that promote remodeling of the extracellular matrix (ECM) in the cervix .

• During pregnancy, the ECM helps maintain cervical firmness and structure, while at term, ANTXR2 promotes ECM remodeling to allow cervical softening and dilation (ripening) .

• Older female mice lacking ANTXR2 were less likely to become pregnant due to structural abnormalities of the uterus, suggesting age-dependent reproductive effects of ANTXR2 function .

How might dysregulation of ANTXR2 contribute to pregnancy complications?

Dysregulation of ANTXR2's role in ECM remodeling could contribute to pregnancy complications:

• Premature cervical ripening: If ANTXR2 inappropriately activates MMPs too early in pregnancy, it could lead to cervical insufficiency and preterm labor .

• Failure to progress in labor: Insufficient ANTXR2 function may prevent proper cervical ripening at term, as seen in knockout mice that were unable to deliver .

Based on these findings, researchers at Columbia formed the Collaborative Cervical Research Group to develop tests for determining the risk of preterm labor in humans by investigating ANTXR2 function and regulation .

What techniques are used to study ANTXR2 protein interactions?

Researchers employ several methods to study ANTXR2 protein interactions:

• Surface plasmon resonance (SPR):

  • Used to measure direct protein-protein interactions with quantitative binding kinetics

  • Performed using systems like BIAcore 2000

  • Can determine association rate constants (ka), dissociation rate constants (kd), and equilibrium dissociation constants (KD)

  • Example: SPR confirmed that the VWA domains of ANTXR1 and ANTXR2 bind to protective antigen (PA) with KD values of 182 ± 64.3 nM and 6.45 ± 1.08 nM, respectively

• Pull-down assays:

  • Used to study interactions between full-length receptors and potential ligands

  • Employs Indigo-Ni Agarose beads or Strep-Tactin coupled sepharose beads

  • Involves incubation with conditioned media containing potential binding partners

  • Detection by immunoblotting after elution

• Cell binding assays:

  • Stable overexpression of full-length receptors in cell lines

  • Treatment with potential ligands followed by immunofluorescence detection

  • Allows visualization of binding at the cellular level

How can ANTXR2 expression be experimentally manipulated in research models?

Several approaches are used to manipulate ANTXR2 expression:

• CRISPRi knockdown:

  • Targets cis-regulatory elements (CREs) affecting ANTXR2 expression

  • Example: Knockdown of CRE3 and CRE4 decreased ANTXR2 expression by 45% and 62%, respectively, in K562 cells

• Genetic knockout models:

  • Generation of mice lacking functional ANTXR2

  • Useful for studying physiological roles in vivo, particularly in reproductive biology

• Cell line overexpression:

  • Stable transfection of cell lines (e.g., HEK293T) to overexpress wild-type or mutant ANTXR2

  • Allows functional studies of receptor activity

• eQTL analysis:

  • Identification of expression quantitative trait loci that affect ANTXR2 expression levels

  • Links genetic variation to expression differences across tissues

How do we reconcile conflicting data on ANTXR2's role as a collagen VI receptor?

There are conflicting reports regarding ANTXR2's role as a collagen VI receptor:

• Negative evidence:

  • Direct protein-protein interaction studies using surface plasmon resonance failed to detect binding between the VWA domain of ANTXR2 and the C5 domain of collagen VI

  • Cell binding assays with ANTXR2-overexpressing cells did not show increased binding to collagen VI or its C5 domain

  • Pull-down assays could not co-precipitate collagen VI with ANTXR2, while control proteins (PA and NG2) showed expected binding

• Proposed indirect mechanisms:

  • ANTXR2 may contribute indirectly to collagen VI biology, possibly by activating proteases located at the plasma membrane

  • Methodological differences between studies might explain discrepancies in reported results

Future research should investigate alternative mechanisms by which ANTXR2 might influence collagen VI function without direct binding, possibly through:

• Regulation of proteolytic processing

• Effects on shared signaling pathways

• Formation of larger protein complexes with additional binding partners

What are the tissue-specific effects of human-specific changes in ANTXR2 expression?

While ANTXR2 downregulation in human immune cells is well-documented, research indicates more complex tissue-specific patterns:

• Immune cells: Consistent 8-fold downregulation across all human samples compared to non-human primates

• Reproductive tissues: Crucial role in cervical remodeling during pregnancy, but evolutionary changes between humans and other species are less clear

• European-specific changes: Evidence of recent positive selection driving decreased expression in multiple tissues affected by anthrax toxins specifically in European populations

Future research directions should include:

• Comparative analysis of ANTXR2 expression and function across a broader range of tissues

• Investigation of tissue-specific regulatory elements affected by human-specific mutations

• Functional consequences of expression differences in various physiological contexts

What statistical approaches are recommended for analyzing evolutionary changes in ANTXR2?

When analyzing evolutionary changes in ANTXR2, researchers should consider:

• Comparative transcriptomic analysis:

  • DESeq2 for differential transcription analysis with FDR correction for multiple testing

  • Wilcoxon rank-sum test for comparing transcription levels across species

• Selection analysis:

  • SweepFinder2 for detecting recent selective sweeps

  • Maximum parsimony approaches for inferring lineage-specific changes

• eQTL identification:

  • Analysis of RNA-seq data to identify expression quantitative trait loci

  • Integration with CRE mapping to understand genetic variation effects

• Multiple testing correction:

  • For data comparison between multiple groups, one-way ANOVA with Dunnett multiple testing correction is recommended

  • P value ≤ 0.05 should be considered significant