Binding Evolution of the Dengue Virus Envelope Against DC-SIGN: A Combined Approach of Phylogenetics and Molecular Dynamics Analyses Over 30 Years of Dengue Virus in Brazil.

The Red Queen Hypothesis (RQH), derived from Lewis Carroll's "Through the Looking-Glass", postulates that organisms must continually adapt in response to each other to maintain relative fitness. Within the context of host-pathogen interactions, the RQH implies an evolutionary arms race, wherein viruses evolve to exploit hosts and hosts evolve to resist viral invasion. This study delves into the dynamics of the RQH in the context of virus-cell interactions, specifically focusing on virus receptors and cell receptors. We observed multiple virus-host systems and noted patterns of co-evolution. As viruses evolved receptor-binding proteins to effectively engage with cell receptors, cells countered by altering their receptor genes. This ongoing mutual adaptation cycle has influenced the molecular intricacies of receptor-ligand interactions. Our data supports the RQH as a driving force behind the diversification and specialization of both viral and host cell receptors. Understanding this co-evolutionary dance offers insights into the unpredictability of emerging viral diseases and potential therapeutic interventions. Future research is crucial to dissect the nuanced molecular changes and the broader ecological consequences of this ever-evolving battle. Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time.

Secondary Sites of the C-type Lectin-Like Fold.

C-type lectins are a large superfamily of proteins involved in a multitude of biological processes. In particular, their involvement in immunity and homeostasis has rendered them attractive targets for diverse therapeutic interventions. They share a characteristic C-type lectin-like domain whose adaptability enables them to bind a broad spectrum of ligands beyond the originally defined canonical Ca2+-dependent carbohydrate binding. Together with variable domain architecture and high-level conformational plasticity, this enables C-type lectins to meet diverse functional demands. Secondary sites provide another layer of regulation and are often intricately linked to functional diversity. Located remote from the canonical primary binding site, secondary sites can accommodate ligands with other physicochemical properties and alter protein dynamics, thus enhancing selectivity and enabling fine-tuning of the biological response. In this review, we outline the structural determinants allowing C-type lectins to perform a large variety of tasks and to accommodate the ligands associated with it. Using the six well-characterized Ca2+-dependent and Ca2+-independent C-type lectin receptors DC-SIGN, langerin, MGL, dectin-1, CLEC-2 and NKG2D as examples, we focus on the characteristics of non-canonical interactions and secondary sites and their potential use in drug discovery endeavors.

Programming multicellular assembly with synthetic cell adhesion molecules.

Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization.

Glycan Conformation in the Heavily Glycosylated Protein, CEACAM1.

Glycans attached to glycoproteins can contribute to stability, mediate interactions with other proteins, and initiate signal transduction. Glycan conformation, which is critical to these processes, is highly variable and often depicted as sampling a multitude of conformers. These conformers can be generated by molecular dynamics simulations, and more inclusively by accelerated molecular dynamics, as well as other extended sampling methods. However, experimental assessments of the contribution that various conformers make to a native ensemble are rare. Here, we use long-range pseudo-contact shifts (PCSs) of NMR resonances from an isotopically labeled glycoprotein to identify preferred conformations of its glycans. The N-terminal domain from human Carcinoembryonic Antigen Cell Adhesion Molecule 1, hCEACAM1-Ig1, was used as the model glycoprotein in this study. It has been engineered to include a lanthanide-ion-binding loop that generates PCSs, as well as a homogeneous set of three 13C-labeled N-glycans. Analysis of the PCSs indicates that preferred glycan conformers have extensive contacts with the protein surface. Factors leading to this preference appear to include interactions between N-acetyl methyls of GlcNAc residues and hydrophobic surface pockets on the protein surface.

Crystal structure of Ankyrin-G in complex with a fragment of Neurofascin reveals binding mechanisms required for integrity of the axon initial segment.

The axon initial segment (AIS) has characteristically dense clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin 186 (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitates generation of an action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly, maintenance, and plasticity remain poorly understood. Here, we report the high-resolution crystal structure of the AnkG ankyrin repeat (ANK repeat) domain in complex with its binding site in the Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays with serial truncation variants, the molecular basis of AnkG-Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and we identify another region required for their high affinity binding. Our structural analysis revealed that ANK repeats form 4 hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with essential Nfasc residues, including F1202, E1204, and Y1212. Moreover, we show disruption of the AnkG-Nfasc complex abolishes Nfasc enrichment at the AIS in cultured mouse hippocampal neurons. Finally, our structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM, a member of the L1 immunoglobulin family proteins including Nfasc, L1CAM, NrCAM, and CHL1, compromise binding with ankyrins. Taken together, these results define the mechanisms underlying AnkG-Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.

Molecular ZIP codes in targeted drug delivery.

The term "molecular ZIP (or area) codes" refers to an originally hypothetical system of cell adhesion molecules that would control cell trafficking in the body. Subsequent discovery of the integrins, cadherins, and other cell adhesion molecules confirmed this hypothesis. The recognition system encompassing integrins and their ligands came particularly close to fulfilling the original ZIP code hypothesis, as multiple integrins with closely related specificities mediate cell adhesion by binding to an RGD or related sequence in various extracellular matrix proteins. Diseased tissues have their own molecular addresses that, although not necessarily involved in cell trafficking, can be made use of in targeted drug delivery. This article discusses the molecular basis of ZIP codes and the extensive effort under way to harness them for drug delivery purposes.

A novel class of Candida glabrata cell wall proteins with ß-helix fold mediates adhesion in clinical isolates.

Candida glabrata is an opportunistic pathogenic yeast frequently causing infections in humans. Though it lacks typical virulence factors such as hyphal development, C. glabrata contains a remarkably large and diverse set of putative wall adhesins that is crucial for its success as pathogen. Here, we present an analysis of putative adhesins from the homology clusters V and VI. First, sequence similarity network analysis revealed relationships between cluster V and VI adhesins and S. cerevisiae haze protective factors (Hpf). Crystal structures of A-regions from cluster VI adhesins Awp1 and Awp3b reveal a parallel right-handed ß-helix domain that is linked to a C-terminal ß-sandwich. Structure solution of the A-region of Awp3b via single wavelength anomalous diffraction phasing revealed the largest known lanthanide cluster with 21 Gd3+ ions. Awp1-A and Awp3b-A show structural similarity to pectate lyases but binding to neither carbohydrates nor Ca2+ was observed. Phenotypic analysis of awp1Δ, awp3Δ, and awp1,3Δ double mutants did also not confirm their role as adhesins. In contrast, deletion mutants of the cluster V adhesin Awp2 in the hyperadhesive clinical isolate PEU382 demonstrated its importance for adhesion to polystyrene or glass, biofilm formation, cell aggregation and other cell surface-related phenotypes. Together with cluster III and VII adhesins our study shows that C. glabrata CBS138 can rely on a set of 42 Awp1-related adhesins with ß-helix/α-crystallin domain architecture for modifying the surface characteristics of its cell wall.

A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

Phosphorylation of human CEACAM1-LF by PKA and GSK3ß promotes its interaction with ß-catenin.

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for ß-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bß but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3ß binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3ß able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of ß-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3ß may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with ß-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.

Role of Nectin­4 protein in cancer (Review).

The Nectin cell adhesion molecule (Nectin) family members are Ca2+­independent immunoglobulin­like cellular adhesion molecules (including Nectins 1­4), involved in cell adhesion via homophilic/heterophilic interplay. In addition, the Nectin family plays a significant role in enhancing cellular viability and movement ability. In contrast to enrichment of Nectins 1­3 in normal tissues, Nectin­4 is particularly overexpressed in a number of tumor types, including breast, lung, urothelial, colorectal, pancreatic and ovarian cancer. Moreover, the upregulation of Nectin­4 is an independent biomarker for overall survival in numerous cancer types. A large number of studies have revealed that high expression of Nectin­4 is closely related to tumor occurrence and development in various cancer types, but the manner in which Nectin­4 protein contributes to the onset and development of these malignancies is yet unknown. The present review summarizes the molecular mechanisms and functions of Nectin­4 protein in the biological processes and current advances with regard to its expression and regulation in various cancer types.

Glycan-Lectin Interactions in Cancer and Viral Infections and How to Disrupt Them.

Glycan-lectin interactions play an essential role in different cellular processes. One of their main functions is involvement in the immune response to pathogens or inflammation. However, cancer cells and viruses have adapted to avail themselves of these interactions. By displaying specific glycosylation structures, they are able to bind to lectins, thus promoting pathogenesis. While glycan-lectin interactions promote tumor progression, metastasis, and/or chemoresistance in cancer, in viral infections they are important for viral entry, release, and/or immune escape. For several years now, a growing number of investigations have been devoted to clarifying the role of glycan-lectin interactions in cancer and viral infections. Various overviews have already summarized and highlighted their findings. In this review, we consider the interactions of the lectins MGL, DC-SIGN, selectins, and galectins in both cancer and viral infections together. A possible transfer of ways to target and disrupt them might lead to new therapeutic approaches in different pathological backgrounds.

Trop2 Forms a Stable Dimer with Significant Structural Differences within the Membrane-Distal Region as Compared to EpCAM.

Trop2 is a cell-surface transmembrane glycoprotein involved in the maintenance of epithelial tissue integrity and is an important carcinoma marker. It shares similar claudin-interaction capacity with its paralogue EpCAM, and both are implicated in signaling triggered by proteolytic cleavage within the ectodomain. However, the cell proliferation-regulating interactions with IGF-1, neuregulin-1, and α5ß1 integrin appear to be Trop2-specific. To illuminate the structural differences between Trop2 and EpCAM, we report the first crystal structure of a Trop2 ectodomain dimer and compare it to the analogous part of EpCAM. While the overall fold of the two proteins is similar, the dimers differ. In Trop2, the inter-subunit contacts are more extensive than in EpCAM, and there are two major differences in the membrane-distal regions. The immunogenic N-terminal domain is in Trop2 almost colinear with the dimer interface plain and consequently more laterally exposed, and the cleft of yet unknown functionality between the two subunits is almost absent. Furthermore, the site of initial signaling-associated proteolytic cleavage in Trop2 is accessible in the dimeric state, while in EpCAM dimer destabilization is required. The structural differences highlight the divergent evolutionary path of the two proteins and pave the way for their structure-based utilization in therapy.

Structure of cell-cell adhesion mediated by the Down syndrome cell adhesion molecule.

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.

Periostin in Allergy and Inflammation.

Matricellular proteins are involved in the crosstalk between cells and their environment and thus play an important role in allergic and inflammatory reactions. Periostin, a matricellular protein, has several documented and multi-faceted roles in health and disease. It is differentially expressed, usually upregulated, in allergic conditions, a variety of inflammatory diseases as well as in cancer and contributes to the development and progression of these diseases. Periostin has also been shown to influence tissue remodelling, fibrosis, regeneration and repair. In allergic reactions periostin is involved in type 2 immunity and can be induced by IL-4 and IL-13 in bronchial cells. A variety of different allergic diseases, among them bronchial asthma and atopic dermatitis (AD), have been shown to be connected to periostin expression. Periostin is commonly expressed in fibroblasts and acts on epithelial cells as well as fibroblasts involving integrin and NF-κB signalling. Also direct signalling between periostin and immune cells has been reported. The deposition of periostin in inflamed, often fibrotic, tissues is further fuelling the inflammatory process. There is increasing evidence that periostin is also expressed by epithelial cells in several of the above-mentioned conditions as well as in cancer. Augmented periostin expression has also been associated with chronic inflammation such as in inflammatory bowel disease (IBD). Periostin can be expressed in a variety of different isoforms, whose functions have not been elucidated yet. This review will discuss potential functions of periostin and its different isoforms in allergy and inflammation.

Dual-Multivalent-Aptamer-Conjugated Nanoprobes for Superefficient Discerning of Single Circulating Tumor Cells in a Microfluidic Chip with Inductively Coupled Plasma Mass Spectrometry Detection.

The efficient recognition of circulating tumor cells (CTCs) with an aptamer probe confers numerous benefits; however, the stability and binding affinity of aptamers are significantly hampered in real biological sample matrices. Inspired by the efficient preying mechanism by multiplex tubing feet and endoskeletons of sea urchins, we engineered a superefficient biomimetic single-CTC recognition platform by conjugating dual-multivalent-aptamers (DMAs) Sgc8 and SYL3C onto AuNPs to form a sea urchin-like nanoprobe (sea urchin-DMA-AuNPs). Aptamers Sgc8 and SYL3C selectively bind with the biomarker proteins PTK7 and EpCAM expressed on the surface of CTCs. CTCs were captured with 100% efficiency, followed by sorting on a specially designed multifunctional microfluidic configuration, integrating a single-CTC separation unit and a hydrodynamic filtrating purification unit. After sorting, background-free analysis of biomarker proteins in single CTCs was undertaken with inductively coupled plasma mass spectrometry by measuring the amount of 197Au isotope in sea urchin-DMA-AuNPs. With respect to a single-aptamer nanoprobe/-interface, the dual-aptamer nanoprobe improves the binding efficiency by more than 200% (Kd < 0.35 nM). The microchip facilitates the recognition of single CTCs with a sorting separation rate of 93.6% at a flow rate of 60 µL min-1, and it exhibits 73.8 ± 5.0% measurement efficiency for single CTCs. The present strategy ensures the manipulation and detection of a single CTC in 100 µL of whole blood within 1 h.

Pathological Roles and Clinical Usefulness of Periostin in Type 2 Inflammation and Pulmonary Fibrosis.

Periostin is known to be a useful biomarker for various diseases. In this article, we focus on allergic diseases and pulmonary fibrosis, for which we and others are now developing detection systems for periostin as a biomarker. Biomarker-based precision medicine in the management of type 2 inflammation and fibrotic diseases since heterogeneity is of utmost importance. Periostin expression is induced by type 2 cytokines (interleukin-4/-13) or transforming growth factor-ß, and plays a vital role in the pathogenesis of allergic inflammation or interstitial lung disease, respectively, andits serum levels are correlated disease severity, prognosis and responsiveness to the treatment. We first summarise the importance of type 2 biomarker and then describe the pathological role of periostin in the development and progression of type 2 allergic inflammation and pulmonary fibrosis. In addition, then, we summarise the recent development of assay methods for periostin detection, and analyse the diseases in which periostin concentration is elevated in serum and local biological fluids and its usefulness as a biomarker. Furthermore, we describe recent findings of periostin as a biomarker in the use of biologics or anti-fibrotic therapy. Finally, we describe the factors that influence the change in periostin concentration under the healthy conditions.

Recent advances in trophoblast cell-surface antigen 2 targeted therapy for solid tumors.

Trophoblast cell-surface antigen 2 (Trop 2) is a transmembrane glycoprotein that is highly expressed in various cancer types with relatively low or no baseline expression in most normal tissues. Its overexpression is associated with tumor growth and poor prognosis; Trop 2 is, therefore, an ideal therapeutic target for epithelial cancers. Several Trop 2 targeted therapeutics have recently been developed for the treatment of cancers, such as anti-Trop 2 antibodies and antibody-drug conjugates (ADCs), as well as Trop 2-specific cell therapy. In particular, the safety and clinical benefit of Trop 2-based ADCs have been demonstrated in clinical trials across multiple tumor types, including those with limited treatment options, such as triple-negative breast cancer, platinum-resistant urothelial cancer, and heavily pretreated non-small cell lung cancer. In this review, we elaborate on recent advances in Trop 2 targeted modalities and provide an overview of novel insights for future developments in this field.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell-like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

Participation of selected soluble cell adhesion molecules and syndecans in formation and development of endometriosis.

OBJECTIVES: Concentrations of soluble ICAM-2, -3, -4 and syndecan-1 and -4 have not yet been marked in the peritoneal fluid of women with endometriosis. The aim of the study was to determine whether these molecules can participate in formation and development of endometriosis. MATERIAL AND METHODS: The study comprised of 80 women at the proliferative phase of the menstrual cycle, aged 21 to 49 years (mean age 31. 3 ± 6. 7 years) undergoing laparoscopy, to determine the causes of primary infertility and to confirm or exclude endometriosis. The study group consisted of 60 women with endometriosis in the pelvis as confirmed by laparoscopy and histopathology. The reference group consisted of 20 women in whom no endometriosis. Concentrations of selected sICAM and syndecans in the peritoneal fluid were determined with the use of ELISA method. RESULTS: Decreased concentrations of sICAM-2 and increased concentrations of sICAM-3, sICAM-4 and syndnecan-1 and -4 were observed in the peritoneal fluid of women with endometriosis and compared with concentrations of this parameter in the reference group (p < 0.0001). Additionally, negative correlation was found between the concentrations of sICAM-3 and sICAM-2 among women with endometriosis. There was no statistically significant correlation between the concentration of sICAM-2 and sICAM-4, sICAM-3 and sICAM-4 and syndecan-1 and syndecan-4 in the examined women. CONCLUSIONS: Changes in concentrations of all the evaluated molecules were observed in the peritoneal fluid in women suffering from endometriosis. Since they have a role in regulation of the immune response, in angiogenesis and apoptosis of the endometrial cells.

Intron-targeted mutagenesis reveals roles for Dscam1 RNA pairing architecture-driven splicing bias in neuronal wiring.

Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. Here, we provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. We generate mutant flies with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D-/- and DscamΔ9D-/-), respectively. In contrast to DscamΔ4D-/-, DscamΔ9D-/- exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. We conclude that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling.