Secondary muscle atrophy resulting from neuronal denervation is a key determinant of poor functional recovery in immune-mediated neuropathies, even after inflammation is resolved. While current immunotherapies effectively limit immune-mediated nerve injury, secondary muscle atrophy as a downstream consequence of denervation is not addressed by existing treatment strategies. Activin type II receptors (ActIIR) are central regulators of skeletal muscle atrophy, and pharmacological inhibition of ActIIR signaling has shown variable efficacy in primary myopathies. Here, we investigated the therapeutic potential of ActIIR inhibition in promoting motor recovery in immune-mediated neuropathies using experimental autoimmune neuritis, a rodent model of Guillain-Barré syndrome. At the onset of the recovery phase, animals received varying doses of a synthesized anti-ActIIR antibody. Motor performance was assessed using a standardized clinical neuritis score, grip strength testing, electrophysiological nerve conduction studies, and balance-beam performance combined with quantitative kinematic gait analysis. High-dose ActIIR antibody treatment significantly improved motor performance during the recovery phase. This improvement was not associated with detectable changes in peripheral nerve immune infiltration, inflammatory cytokine expression, or myelination, but correlated with preservation of muscle fiber size. Consistent with this, muscle proteomic and targeted transcriptional analyses revealed attenuation of FoxO-dependent atrophy programs and reduced expression of the E3 ubiquitin ligases Atrogin-1 and MuRF1. Together, these findings suggest that ActIIR inhibition limits secondary muscle atrophy and enhances motor recovery in autoimmune neuritis. Targeting ActIIR signaling may therefore represent a promising muscle-directed adjunct strategy to improve motor outcomes in immune-mediated neuropathies.

Abstract Orthoebolaviruses such as Ebola virus (EBOV), Sudan virus (SUDV) and Bundibugyo virus (BDBV) can cause severe disease with high case-fatality rates. While licensed EBOV vaccines and therapeutic antibodies protect against EBOV infection, no single monoclonal antibody currently provides broad protection across multiple orthoebolaviruses. Here, we analyzed the humoral immune response of an rVSV-EBOV vaccinee to identify pan- orthoebolavirus -neutralizing antibodies. Using BDBV- and SUDV-glycoproteins for single B cell-sorting, we identified B10, which neutralized authentic EBOV and SUDV, with potent activity against SUDV compared with established cross-reactive antibodies. Structural analysis mapped antibody B10 binding to the pan- orthoebolavirus conserved GP2-stalk/HR2 region, associated with asymmetric trimer destabilization and spike opening. In vivo, B10 showed significant prophylactic efficacy in an EBOV mouse model and partial protection with antiviral activity in a SUDV mouse model. Together, these findings demonstrate that rVSV-EBOV vaccination induced the development of a broadly orthoebolavirus-neutralizing antibody that holds exeptional therapeutic potential.

ABSTRACT Objectives To evaluate serum IgG responses against Porphyromonas gingivalis gingipains RgpB and Kgp in patients with stage III and IV periodontitis and examine associations with clinical periodontal parameters. Materials and Methods This exploratory cross‐sectional study included participants with advanced periodontitis (Stage III and IV). Clinical periodontal parameters (number of teeth, bleeding on probing in percent [BoP%], probing pocket depth [PPD], bone loss‐to‐age ratio, periodontal inflamed surface area [PISA] per tooth) were recorded. Blood samples were collected and serum IgG responses against recombinant RgpB and Kgp were analyzed. Serum IgG signals were quantified using enzyme‐linked immunosorbent assay (ELISA). Results All 42 participants (mean age 62 years) exhibited measurable serum IgG responses against P. gingivalis gingipains. IgG signals (optical density) ranged from 0.13 to 1.46 for RgpB and 0.34 to 1.51 for Kgp. RgpB‐specific IgG levels were higher in Stage IV versus Stage III periodontitis (p = 0.023), whereas Kgp‐specific IgG levels showed no significant difference between stages (p = 0.249). Anti‐RgpB IgG levels correlated positively with age (ρ = 0.362, p = 0.019) and inversely with the number of teeth (ρ = −0.372, p = 0.015), whereas anti‐Kgp IgG showed no statistically significant correlations with the assessed clinical parameters. Conclusions Serum IgG binding signals to RgpB and Kgp were measurable by ELISA and showed substantial heterogeneity among patients with advanced periodontitis, with considerable overlap between disease stages. Anti‐RgpB IgG was associated with age and tooth loss, whereas inflammatory measures showed no significant correlations and anti‐Kgp IgG showed only weak trend‐level associations. Overall, total gingipain‐specific IgG appears to reflect cumulative periodontal disease burden and exposure history more clearly than current inflammatory activity in this cohort, with limited value as a standalone marker for differentiating Stage III and Stage IV periodontitis.

Alveolar growth and repair are central processes in development and chronic lung disease, such as bronchopulmonary dysplasia (BPD), a neonatal lung disease without curative therapy. Alveolar epithelial type 2 (AT2) cells are the endogenous progenitor pool giving rise to alveolar epithelial type 1 cells and promoting alveolar repair. Since netrin-1, a regulator of cell homeostasis and stemness, has been linked to lung diseases, we now investigated its signaling and function in AT2 cells in a hyperoxia-based model of BPD and in lungs of infants with BPD. First, we demonstrated that prolonged hyperoxia reduced both netrin-1 and its receptor Unc5b in neonatal mouse lungs and in primary AT2 cells. Second, ex vivo studies using precision-cut lung slices (PCLS) and primary murine AT2 cell culture showed that netrin-1 regulates AT2 cell survival and the expression of Krüppel-like factor 4 (Klf4) through Unc5b, a transcription factor regulating cell survival. Third, single-cell and bulk transcriptomic analysis, as well as proximity-dependent biotin identification assay, showed Klf4 to be upregulated in AT2 cells during alveolarization, downstream of netrin-1, and to regulate AT2 cell survival. In vivo, Klf4 gene expression and protein abundance was significantly reduced in total lung homogenates and in AT2 cells of neonatal mice exposed to hyperoxia. Finally, KLF4+ cells, KLF4+ epithelial, and specifically KLF4+ AT2 cells were reduced in clinical BPD. In summary, our data identify a novel netrin-1-Unc5b-Klf4 axis in AT2 cells that is disrupted in BPD and could offer a novel target for endogenous alveolar repair.

Given that head and neck squamous cell carcinoma (HNSCC) patients have poor survival outcomes, a better understanding of the therapeutic benefits of ionizing irradiation (IR), the major treatment modality besides surgery, is needed. A confounding factor is the immunosuppressive tumor microenvironment determined by tenascin-C (TNC), a highly abundant extracellular matrix molecule upregulated by IR. We investigated the roles of TNC on radio-induced tumor regression in a murine oral HNSCC model expressing or lacking TNC. While tumors in a TNC-expressing host were radiosensitive, they were radioresistant in TNC genetically depleted mice. We identified fibroblast reticular cells (FRCs) as critical regulators. TNC plays a compartmentalized and dual role in regulating tumor radiosensitivity with a detrimental role in the tumor stroma opposed to an essential role in the tumor-draining lymph nodes. This is relevant as a high FRC signature and high TNC levels together correlate with shorter HNSCC patient survival. TNC-expressing FRCs may be an excellent novel target to improve radiotherapy-induced tumor eradication, as our TNC targeting MAREMO peptide reduced tumor cell numbers and plasticity upon IR.

The extracellular matrix provides a crucial tissue-specific signaling hub and structural scaffold that transduces biomechanical force load into cellular responses. While this is especially important in the musculoskeletal joint, the spatial organization of extracellular matrix (ECM) assemblies has been poorly investigated. Dense supra-structures, inaccessible epitopes and complex antigen retrieval negatively affect the 3D-visualization of ECM-scaffolds in connective tissues. We have developed multiplex ECM immuno-staining techniques that now permit mapping of the entire musculoskeletal ECM with embedded cells during skeletal growth and repair. Specifically, we show that osteoblasts deposit a bony ECM on the cartilaginous template in the hypertrophic growth plate, creating a unique ECM composite that likely confers both stiffness and flexibility to neonatal bone. We demonstrate that tendon collagen I fibrils anchor along a thin, continuous tidemark at the lateral cartilaginous surface, mechanically coupling cartilage and tendon matrices at contact sites. Finally, we reveal muscle-derived laminin γ1(+) vascular basement membrane infiltration of the cartilaginous fracture callus ECM, likely facilitating nutrient delivery and structural stabilization during skeletal repair. These advances provide insight into the complexity of the ECM and its interplay with embedded cell clusters at previously unattainable spatial resolution and biological context during skeletal growth and repair.

The molecular mechanisms that drive (lymph-)angiogenesis are crucial to understand diseases, such as lymphedema, that are caused due to malformations of the lymphatic vasculature. Recently, an interaction between the secreted protein Svep1, a key regulator in lymphangiogenesis, and the transmembrane receptor Tie1 was shown in zebrafish, human, and mice. Here, guided by in silico AlphaFold-multimer structure predictions of SVEP1 complexes, we assert with protein binding studies that the human CCP20 domain is the primary binding site for TIE1. We further demonstrate that SVEP1 mediates strong binding of ANG2 and TIE1, and that combined stimulation of hdLECs with SVEP1 and ANG2 leads to phosphorylation of TIE1. TIE1 activation by SVEP1 and ANG2 enables downstream signaling and, in turn, potentiates nuclear exclusion of FOXO1 and phosphorylation of AKT compared to SVEP1 or ANG2 alone. We present a model in which ANG1/2 dimers bind to both SVEP1 and TIE1, resulting in the recruitment of multiple TIE1 receptor molecules to a multimeric complex at the cell membrane, potentially amplifying its signaling capacity.

The endoplasmic reticulum (ER) orchestrates the secretion of extracellular matrix (ECM) proteins, many of which exceed the size of conventional transport vesicles and therefore require specialized export machinery. TANGO1, encoded by MIA3, organizes ER exit sites for bulky cargo export, yet the molecular basis of cargo recognition and its relationship to the collagen-specific chaperone HSP47 remain unclear. Through quantitative secretome profiling of TANGO1- and HSP47-deficient fibroblasts, structural modeling, and binding analyses, we show that TANGO1 directly and selectively recognizes a defined subset of ECM proteins, including specific collagen and fibrillin isoforms, through a conserved tyrosine residue in its luminal MOTH domain, independently of HSP47. Novel MIA3 variants identified in individuals with previously undescribed skeletal dysplasia disrupt this cargo-binding interface, leading to selective intracellular retention of ECM proteins. These findings identify direct cargo recognition, rather than ER exit-site assembly, as the primary molecular defect underlying MIA3/TANGO1-associated skeletal dysplasia. More broadly, our work establishes TANGO1 as a selective ECM cargo receptor that functions independently of HSP47, providing a new framework for understanding bulky cargo selection at the ER.

Immune memory plays a critical role in the development of durable antimicrobial immune responses. How precisely mRNA vaccines train innate immune cells to shape protective host defense mechanisms remains unknown. Here we show that SARS-CoV-2 mRNA vaccination significantly establishes histone H3 lysine 27 acetylation (H3K27ac) at promoters of human monocyte-derived macrophages, suggesting epigenetic memory. However, we found that two consecutive vaccinations were required for the persistence of H3K27ac, which matched with pro-inflammatory innate immune-associated transcriptional changes and antigen-mediated cytokine secretion. H3K27ac at promoter regions were preserved for six months and a single mRNA booster vaccine potently restored their levels and release of macrophage-derived cytokines. Interestingly, we found that H3K27ac at promoters is enriched for G-quadruplex DNA secondary structure-forming sequences in macrophage-derived nucleosome-depleted regions, linking epigenetic memory to nucleic acid structure. Collectively, these findings reveal that mRNA vaccines induce a highly dynamic and persistent training of innate immune cells enabling a sustained pro-inflammatory immune response.

The myotendinous junction (MTJ) is a weak link in the musculoskeletal system. Here, we isolated the tips of single myofibres from healthy (non-injured) human hamstring muscles for confocal microscopy (n=6) and undertook RNAscope in situ hybridisation (n=6) to gain insight into the profiles of cells and myonuclei in this region, in a fibre type manner. A marked presence of mononuclear cells was observed coating the myofibre tips (confirmed by serial block face scanning electron microscopy and cryosection immunofluorescence), with higher numbers for type I (median 29; range 16-63) than type II (16; 9-23) myofibres (P<0.05). The number of these cells expressing COL22A1 was comparable between fibre types. Myonuclear number and density gradually increased from the myofibre proper towards the tip for both fibre types (P<0.05). COL22A1 was expressed by similar proportions of myonuclei in type I (median 26%; range 13-56) and type II (19%; 3-67) myofibre tips. 70% of the COL22A1-positive nuclei in the MTJ region were myonuclei, and the remaining 30% were MTJ cells. This insight refines our fundamental understanding of the human MTJ at the cell and structural levels.

Heterotopic ossification of tendons and ligaments causes pain and dysfunction, significantly reducing quality of life. However, its underlying mechanisms remain elusive. In addition to injury, tissue organization and stiffness have been implicated in heterotopic ossification. Collagen XII, a member of the fibril-associated collagens with interrupted triple helices (FACIT) family, plays a crucial role in maintaining the structural integrity and function of tendons and ligaments. Its deficiency alters tissue stiffness and predisposes ligaments to rupture. In this study, we investigated whether collagen XII contributes to the development of heterotopic ossification. Three-dimensional microcomputed tomography (3D-μCT) and X-ray analyses revealed heterotopic bone formation in the knee and ankle ligaments, but not in tendons, of Col12a1-deficient mice, with a 100 % incidence in mice older than 19 weeks. Histological analysis showed the presence of Alcian blue- and Toluidine blue-positive fibrochondrocyte-like cells in Col12a1-deficient ligaments, which were subsequently replaced by bone tissue, as indicated by Alizarin red staining. Real-time qPCR analysis of knee ligaments demonstrated a slight increase in chondrogenic markers and a significant upregulation of osteogenic markers in Col12a1-deficient mice compared with wild-type controls. In vitro chondrogenesis and osteogenesis assays using primary tenocytes from wild-type and Col12a1-deficient mice revealed that collagen XII deficiency enhanced osteogenic potential, whereas chondrogenic potential remained comparable. Our findings indicate that collagen XII deficiency specifically induces heterotopic bone formation in knee and ankle ligaments, occurring via fibrochondrocytes rather than through endochondral or intramembranous ossification.

RATIONALE: Lung matrix remodeling by activated fibroblasts is a key pathomechanism of bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF). Epigenetic modifiers could contribute to the onset and progression of lung fibrosis. We recently identified Krüppel-like factor 4 (Klf4) as a key transcription factor in fibroblast cellular activity through an EP300-related interactome. Hence, we now studied if the Klf4-Ep300 axis is a regulator of the acetylome and of chromatin remodeling in fibroblasts in BPD and IPF, favoring thereby lung fibrosis.METHODS: (i) a neonatal murine exposed to hyperoxia (HYX) or normoxia (NOX) until postnatal day (P)14 or P28, (ii) transgenic mice with an ACTA2+ cell-specific deletion of Klf4, (iii) primary neonatal murine and human lung fibroblasts from lungs with IPF and control, and (iv) lungs from patients with BPD or IPF vs control. RESULTS: HYX caused fibrotic lung remodeling in neonatal hyperoxia-exposed mice that was associated with an active TGFβ signaling and reduced Klf4 abundance in proliferative ACTA2+ cells. These findings were related to changes in global and posttranslational protein acetylation as well as to an activation of EP300 in ACTA2+ cells in vivo and in cultured lung fibroblasts, that results in an inactivation of FoxO1. Transgenic mice with ACTA2+ cell-specific ablation of Klf4 exhibited higher matrix remodeling with increased number ACTA2+ cells as well as activation of EP300 and altered histone acetylation. Next, we found Klf4 to regulate proliferation, migration and differentiation in primary neonatal murine and in human lung fibroblasts. Chip-Seq, biotinylation identication assay (BioID), and EP300 inhibition supported a functional role of Klf4 as a key regulator of the global acetylome and FoxO1 acetylation. In addition, we performed ATAC-seq of murine lung fibroblasts from transgenic mice with ACTA2+ cell-specific ablation of Klf4 and from human donor fibroblasts with Klf4 knockdown. Deep epigenetic profiling uncovered a shifted transcription factor footprint favoring FoxO signaling and fibrosis following the loss of Klf4, along with a marked converging epigenetic profile with human IPF-fibroblasts. Finally, human lungs with BPD exhibited significantly reduced abundance of Klf4+ ACTA2+ and an increase in ACTA2+ cells. Similarly, fibrotic foci of IPF lungs showed loss of Klf4. Conversely, Klf4 overexpression exhibited an anti-proliferative and pro-apoptotic effect in IPF lung fibroblasts.CONCLUSION: Our study reveals a Klf4-Ep300 axis as a novel epigenetic modifier of chromatin architecture in lung fibroblasts through modulation of the acetylome, favouring thereby fibrosis and offering a possible therapeutic target for severe BPD and IPF.

Bone morphogenetic protein 10 (BMP-10) is crucial for endothelial cell signaling via activin receptor-like kinase 1 (ALK1), a pathway central to vascular homeostasis and angiogenesis. Dysregulated BMP-10 signaling contributes to cardiovascular diseases and cancer, highlighting the need to control ALK1-mediated endothelial responses to BMP-10 for therapeutic development. BMP-10 biosynthesis involves processing by proprotein convertases (PPCs) resulting in a non-covalently associated prodomain-growth factor (PD-GF) complex (CPLX), similar to other TGF-β superfamily ligands. However, the molecular requirements for BMP-10 bioactivity remain unclear. We investigated how PPC processing impacts BMP-10 structure, bioactivity, and its interaction with the extracellular matrix (ECM) protein fibrillin-1. Molecular dynamics simulations post-in silico cleavage of the BMP-10 dimer model as well as negative staining and transmission electron microscopy (TEM) revealed that PD processing increases BMP-10 flexibility converting it from a latent wide-angle conformation to a bioactive CPLX which can adopt a V-shape with tighter angle. Only processed BMP-10 demonstrated high potency in HUVEC and C2C12 cells and robust binding to immobilized BMP receptors. Circular dichroism and interaction studies revealed that the N-terminal region of the BMP-10 PD is rich in alpha-helical content, which is essential for efficient complexation with the BMP-10 GF. Binding studies and TEM analyses showed that only the processed BMP-10 CPLX interacts with the N-terminal region of fibrillin-1, causing a conformational change that renders it into a closed ring-shaped conformation. These findings suggest that PD processing induces specific folding events at the PD-GF interface, which is critical for BMP-10 bioactivity and its targeting to the ECM.

Collagen VI is a heterotrimeric, ubiquitously expressed microfibrillar collagen with a complex intracellular and extracellular assembly process. In addition to a short collagenous region, it is primarily composed of von Willebrand factor A (VWA) domains. Notably, only the C-terminal end of the α3 chain contains other domain types, including a Kunitz-like C5 domain, which has been reported to be necessary for microfibril formation, to function as a matrikine and exhibit biomarker properties. This region of the α3 chain undergoes proteolytic processing, with cleavage sites identified for proprotein convertases, matrix metalloproteinases (MMPs), and bone morphogenetic protein 1 (BMP1). Cleavage by furin-like convertases results in the generation of a mature collagen VI α3 chain lacking its 70 kDa C2-C5 domains. Here, we provide the first characterization of the functional significance of the furin-like cleavage site, demonstrating that while it is constitutively used, it is not essential for collagen VI assembly, microfibril formation, or skeletal muscle function under physiological conditions, likely due to the presence of redundant cleavage sites. We also present an initial characterization of the biological activity of the released fragments on myoblast cultures showing that they do not affect C2C12 myoblast behaviour or differentiation. These findings deepen our understanding of α3 chain processing and highlight its potential significance for collagen VI assembly and function, including the generation of peptides with potential biomarker and biological activity properties.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers due to late diagnosis and poor therapeutic efficiency. Throughout PDAC progression, a dense extracellular matrix (ECM) is deposited around neoplastic cells and accompanies tumor development and aggressiveness. This significant stroma, both in terms of quantity and through its impact on tumor cells, is now considered as a target for innovative therapies to improve patient survival. Among ECM proteins, Tenascins (TNs) are a family of four glycoproteins (TNC, TNW, TNR and TNX) sharing a common modular structure, but exhibiting different expression patterns and functions depending on the physiological and physio-pathological contexts. In PDAC, TNC is up-regulated and is considered as a pro-tumorigenic actor, whereas TNX role remains to be elucidated. Herein, we demonstrated that unlike TNC, TNX is drastically decreased in PDAC, and that this loss is correlated with reduced patient survival. Dysregulation of the TNX/TNC balance is attributable to Transforming Growth Factor-beta (TGF-β) upregulation during pancreatic carcinogenesis. Interestingly, we found that TNX is first highly deposited in low grade lesions before being clearly decreased in later stages suggesting an elaborate stromal remodelling during PDAC development. Finally, low TNX and high TNC deposition around precursor lesions are correlated with increased preneoplastic cell proliferation. Altogether, our results (i) demonstrate the importance of Tenascin ratios during pancreatic carcinogenesis, (ii) suggest an anti-proliferative role for TNX unlike its pro-tumoral counterpart, TNC and (iii) underscore TNX and TNC as valuable targets for the development of new drugs for pancreatic cancer and/or solid tumor treatment. ### Competing Interest Statement The authors have declared no competing interest.

BackgroundThe role of vitamin D in Coronavirus Disease 2019 (COVID-19) outcomes remains debated, but emerging evidence suggests it may enhance recovery by strengthening immune responses. Vitamin D upregulates LL-37, an antimicrobial peptide with broad antiviral activity, including potential benefits against SARS-CoV-2. LL-37’s interactions with viral proteins, however, remain incompletely understood.MethodsWe investigated LL-37’s interactions with the SARS-CoV-2 Spike glycoprotein and the accessory proteins ORF7a and ORF8 using surface plasmon resonance and negative-stain electron microscopy. These approaches were employed to assess LL-37’s binding capabilities and potential impact on viral infectivity.ResultsLL-37 bound multiple domains of the Spike protein and inhibited its interaction with the human angiotensin-converting enzyme 2 (hACE2) receptor in vitro. Up to seven LL-37 molecules were observed surrounding Spike, forming a halo-like structure that may block receptor engagement. LL-37 also bound to ORF7a and ORF8, potentially impairing their ability to disrupt host cell processes. Notably, LL-37’s interaction with ORF7a may prevent degradation of SNAP29, restoring autophagy and promoting viral clearance.ConclusionsLL-37 disrupts key viral-host interactions by binding to Spike, ORF7a, and ORF8, thereby reducing SARS-CoV-2 infectivity. These findings highlight LL-37’s potential as a therapeutic agent in COVID-19 and provide mechanistic insight into its antiviral actions.

RATIONALE:Mechanical ventilation is a life-saving treatment for preterm infants that often leads to bronchopulmonary dysplasia (BPD). We previously demonstrated a reduced number of alveolar epithelial cells with a depletion of alveolar epithelial type 2 cells (AT2) in lungs of infants with BPD. OBJECTIVE:To investigate and target the mechanisms by which mechanical ventilation causes an arrest of alveolarisation. METHODS:Experimental mouse model of neonatal ventilation-induced lung injury (VILI) in wild-type mice, Il6-null mice, and pharmacological inhibition of interleukin (IL)-6 and endothelin receptors. Complementary, precision-cut lung slices (PCLS) and primary cells were analysed. Moreover, lungs of infants with BPD were studied. RESULTS:Mechanical ventilation leads to an AT2 depletion and arrest of alveolar growth. Transcriptomic profiling, measurement of gene and protein expression, immunofluorescent staining as well as cell culture studies identified an IL-6-mediated expression of Endothelin-1 (Edn1) and a nuclear sequestration of the antiproliferative transcription factor FoxO1 in AT2. These findings were confirmed using murine PCLS, lung epithelial cells and transgenic mice with inducible constitutive active FoxO1. In vivo, Il6-null mice and pharmacological inhibition of IL-6 or endothelin A and B receptors prevented nuclear sequestration of FoxO1, thereby enabling lung growth of newborn mice exposed to mechanical ventilation. CONCLUSION:Mechanical ventilation causes an arrest of alveolarisation in newborn mice through an IL-6-mediated activation of Edn1 signalling and nuclear sequestration of FoxO1 in AT2. Thus, this study provides rationale for considering pharmacological inhibition of IL-6 and/or endothelin receptors as a therapeutic strategy for preterm newborns at risk of VILI-associated lung growth arrest.

Ca2+ is a highly abundant ion involved in numerous biological processes, particularly in multicellular eukaryotic organisms where it exerts many of these functions through interactions with Ca2+ binding proteins. The laminin N-terminal (LN) domain is found in members of the laminin and netrin protein families where it plays a critical role in the function of these proteins. The LN domain of laminins and netrins is a Ca2+ binding domain and in many cases requires Ca2+ to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca2+ interaction combining structural, computational, bioinformatics, and biophysical techniques. By combining computational and bioinformatic techniques with x-ray crystallography we explore the molecular basis of the LN domain Ca2+ interaction and identify a conserved sequence present in Ca2+ binding LN domains. These findings enable a sequence-based prediction of LN domain Ca2+ binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca2+ interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca2+, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca2+ binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca2+ dynamics within the context of the LN domain.