Recombinant Human Ninjurin-2 (NINJ2)

What is the structure and membrane topology of human Ninjurin-2?

Ninjurin-2 (NINJ2) is a 142 amino acid homophilic cell adhesion molecule with an unusual membrane orientation. It consists of a 65 amino acid N-terminal extracellular domain (ECD) (aa 1-65) containing a phosphorylation site at Ser3, followed by a first transmembrane segment, a cytoplasmic region, a second transmembrane segment, and a C-terminal extracellular domain (aa 128-142) . Unlike many other membrane proteins, NINJ2 does not contain N-linked glycosylation sites or a signal sequence . The transmembrane domains represent the most highly conserved regions between NINJ1 and NINJ2 . The NINJ2 gene is located on human chromosome 12p13 and contains three introns, with the intron locations precisely conserved between NINJ1 and NINJ2 .

How does NINJ2 compare structurally and functionally with its paralog NINJ1?

While NINJ2 shares 55% protein identity with human NINJ1, there are critical structural and functional differences:

• NINJ2 (142 aa) is shorter than NINJ1 (152 aa) with a major insertion near the N-terminus in NINJ1

• NINJ2 lacks the homophilic adhesive motif present in residues 26-37 of NINJ1

• Both proteins can assemble into linear filaments that bind strongly to lipids, but NINJ2 filaments have an intrinsic curvature, while NINJ1 filaments are straighter

• The root mean square deviation (RMSD) for the 101 pairs of C-alpha atoms between NINJ1 and NINJ2 is measured to be 0.792 Å, indicating high structural similarity of individual subunits

• Despite their similarities, NINJ1 and NINJ2 do not interact with each other (they engage in homophilic but not heterophilic interactions)

• NINJ1 can mediate plasma membrane rupture (PMR), while NINJ2 cannot due to its intrinsic filament curvature

What is the tissue distribution and expression pattern of NINJ2?

NINJ2 shows a distinct tissue-specific expression pattern:

• Highest expression in bone marrow, followed by peripheral leukocytes, lung, and lymph nodes

• In the peripheral nervous system, NINJ2 is constitutively expressed in mature sensory and enteric neurons but not in glial cells or in autonomic ganglia

• In the central nervous system, NINJ2 is expressed in radial glial cells but not in neurons

• In the kidney, NINJ2 is specifically detected in the glomeruli

• NINJ2 expression is dramatically elevated in differentiated postmitotic neurons during development

• Unlike NINJ1, which is ubiquitously expressed in epithelial tissues such as breast, liver, and spleen, NINJ2 is predominantly expressed in hematopoietic and lymphatic tissues

What are the key biological functions of NINJ2?

NINJ2 serves several important biological roles:

• Cell adhesion: Functions as a homophilic adhesion molecule mediating cell-to-cell interactions through its extracellular domains

• Nerve regeneration: Upregulated in Schwann cells surrounding the distal segment of injured nerves with a time course similar to that of NINJ1, neural CAM, and L1

• Neurite outgrowth: Promotes neurite outgrowth from primary cultured dorsal root ganglion neurons, presumably via homophilic cellular interactions

• p53 regulation: Forms a feedback loop with p53 where NINJ2 is induced by p53 and in turn regulates p53 expression via mRNA translation

• Inflammation: In endothelial cells, NINJ2 activates the TLR4 signaling pathway and promotes inflammation

How is NINJ2 implicated in cardiovascular diseases?

Multiple studies have identified associations between NINJ2 polymorphisms and cardiovascular diseases:

• Coronary Heart Disease (CHD):

  • A case-control study with 499 CHD cases and 505 controls found that rs118050317 significantly increased CHD risk in people aged more than 60 years and in women

  • rs118050317 and rs7307242 showed strong relationships with hypertension risk in CHD patients

  • rs75750647 increased diabetes risk in CHD patients under multiple genetic models, while rs10849390 exhibited protective effects against diabetes in CHD patients

• Stroke:

  • Multiple studies have linked NINJ2 gene polymorphisms with ischemic stroke risk

  • In endothelial cells, NINJ2 activates the TLR4 signaling pathway and promotes inflammation, potentially contributing to vascular pathology

• Other vascular conditions:

  • The NINJ2 gene is located on human chromosome 12p13, a region linked to several disorders including inflammatory bowel disease and acrocallosal syndrome

  • Protein-protein interaction network and functional enrichment analyses suggest NINJ2 plays a significant role in the pathogenesis and progression of CHD

What is the role of NINJ2 in p53-dependent growth regulation?

NINJ2 participates in a complex feedback loop with p53 that has context-dependent effects:

• NINJ2 induction by p53:

  • NINJ2 is induced by DNA damage in a p53-dependent manner

  • p53 directly binds to a p53-responsive element in the NINJ2 promoter, with binding increased following treatment with doxorubicin

• Regulation of p53 by NINJ2:

  • NINJ2 regulates p53 expression via mRNA translation

  • Loss of NINJ2 promotes p53 expression via enhanced mRNA translation

• Opposing effects based on p53 status:

  • In wild-type p53-expressing cells (MCF7, Molt4):

    • Loss of NINJ2 inhibits cell growth and leads to growth suppression

    • NINJ2 deficiency causes premature senescence in mouse embryo fibroblasts (MEFs) in a wild-type p53-dependent manner

  • In mutant p53-expressing cells (MIA-PaCa2):

    • Loss of NINJ2 promotes cell growth and migration

This differential effect based on p53 status suggests that NINJ2 could potentially be a therapeutic target, with effects dependent on the p53 status of the target tissue .

What are the recommended methods for reconstituting and handling recombinant NINJ2?

Based on commercial recombinant NINJ2 protein information:

Reconstitution:

• Recombinant Human NINJ2 (Met1-Thr65, with a C-terminal 6-His tag) is typically provided as a lyophilized preparation from a 0.2 μm filtered solution in PBS

• It should be reconstituted at a concentration of 250 μg/mL in PBS

Storage:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• The product is shipped at ambient temperature but should be stored immediately at the recommended temperature upon receipt

Carrier considerations:

• Recombinant NINJ2 is available with or without a carrier protein (typically Bovine Serum Albumin, BSA)

• Adding a carrier protein enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations

• For cell or tissue culture applications or as an ELISA standard, the version with BSA is generally advisable

• For applications where BSA could interfere, the carrier-free version is recommended

What experimental models are suitable for studying NINJ2 function?

Several experimental models have been successfully used to study NINJ2:

• Cell culture models:

  • Wild-type p53-containing cell lines: MCF7 (human breast cancer) and Molt4 (human T lymphoblast)

  • Mutant p53-containing cell lines: MIA-PaCa2

  • Jurkat cells for adhesion assays

  • Mouse embryo fibroblasts (MEFs) for studying premature senescence

• Genetic manipulation approaches:

  • siRNA-mediated knockdown of NINJ2

  • p53-knockout cell lines for studying p53-dependent aspects of NINJ2 function

  • Comparison studies between NINJ1 and NINJ2 expression systems

• Structural analysis methods:

  • CryoEM for studying NINJ2 filament formation and lipid interactions

  • Negative-staining EM for visualization of protein assemblies

• Functional assays:

  • Colony formation assays to assess cell growth effects

  • ChIP assays to study p53 binding to the NINJ2 promoter

  • Neurite outgrowth assays with primary cultured dorsal root ganglion neurons

  • Cell adhesion assays using homophilic binding properties

What is the molecular mechanism behind NINJ2's inability to mediate plasma membrane rupture compared to NINJ1?

The differential ability to mediate plasma membrane rupture (PMR) between NINJ1 and NINJ2 has been elucidated through structural biology:

• Filament structure differences:

  • Both NINJ1 and NINJ2 assemble into linear filaments that bind strongly to lipids on one side but are water-soluble on the other side

  • NINJ1 filaments are relatively straight, allowing them to wrap around membrane blebs

  • NINJ2 filaments have an intrinsic curvature, forming a spring-shaped assembly with a diameter of approximately 45 nm

  • This fixed curvature prevents NINJ2 from properly wrapping around membrane blebs to induce PMR

• Lipid binding differences:

  • NINJ2 binds more strongly to cholesterol at the inner leaflet of the lipid bilayer, which is responsible for the curving of the NINJ2 filament

  • NINJ1 has stronger lipid binding at the outer leaflet, contributing to its capability of mediating PMR

  • The NINJ1 filament appears slightly skinnier than the NINJ2 filament, as if one central layer of lipids present in the NINJ2 filament were missing in the NINJ1 filament

  • Inner leaflet lipid densities in NINJ1 appear to be more discrete compared to those in NINJ2

• Experimental evidence:

  • Membrane bleb-like particles can be directly observed in negative-staining EM images of purified NINJ1 samples but not in NINJ2 samples

  • Single-layer NINJ1 filaments exhibit considerable lateral flexibility, enabling them to wrap around membrane blebs, whereas the vertical flexibility of both filaments is very limited

How can researchers investigate the homophilic binding mechanisms of NINJ2?

To study NINJ2's homophilic binding properties, researchers can employ several approaches:

• Cell aggregation assays:

  • Express NINJ2 in cell lines like Jurkat cells that normally don't aggregate

  • Mix differentially labeled cells (e.g., red and green fluorescent dyes) to observe homophilic binding

  • Quantify the number of cells in each aggregate to measure binding strength

  • Compare with NINJ1-expressing cells and test for heterophilic interactions by mixing NINJ1 and NINJ2 expressing cells

• Structure-function analysis:

  • Generate truncated versions of NINJ2 to identify critical domains for homophilic binding

  • Create chimeric proteins between NINJ1 and NINJ2 to determine which regions confer specific binding properties

  • Perform site-directed mutagenesis of conserved residues to identify amino acids critical for binding

• Biophysical techniques:

  • Surface plasmon resonance to measure binding kinetics

  • Atomic force microscopy to visualize and quantify binding strength

  • Förster resonance energy transfer (FRET) to detect protein-protein interactions

• Functional assays:

  • Neurite outgrowth assays with primary cultured dorsal root ganglion neurons to assess the functional consequences of homophilic binding

  • Wound healing assays to evaluate how NINJ2-mediated adhesion affects cell migration