DPY19L3 Antibody

What is DPY19L3 and why is it significant for cancer research?

DPY19L3 is a human C-mannosyltransferase that catalyzes the addition of mannose to specific tryptophan residues in proteins through C-mannosylation, a rare type of glycosylation. Recent research has demonstrated that DPY19L3 promotes vasculogenic mimicry (VM) and cell proliferation in cancer cells, including human fibrosarcoma (HT1080) and breast cancer (MDA-MB-231) cell lines . DPY19L3 knockout in HT1080 cells significantly inhibits network formation on Matrigel and reduces proliferation, suggesting its potential as a molecular target for cancer therapy . Additionally, DPY19L3 has been identified as the C-mannosyltransferase of R-spondin1 (Rspo1), a protein that enhances Wnt signaling crucial for embryonic development and several cancers .

Why are DPY19L3 antibodies challenging to develop and validate?

The development of reliable DPY19L3 antibodies presents significant challenges due to several factors:

• Transmembrane topology: DPY19L3 is a multipass transmembrane protein with a complex structure comprising 11 transmembrane regions and two re-entrant loops . This complexity makes it difficult to identify accessible epitopes for antibody recognition.

• Limited immunogenic regions: The topological analysis reveals that DPY19L3 has its N-terminal facing the cytoplasm and C-terminal in the ER lumen , limiting the selection of unique, accessible epitopes.

• Post-translational modifications: DPY19L3 undergoes N-glycosylation at specific sites (Asn 118 and Asn 704) , which may mask potential epitopes or create steric hindrance for antibody binding.

• Isoform variation: The existence of splice variants like isoform2, which lacks the C-terminal luminal region , complicates the development of antibodies that can reliably detect all relevant forms of the protein.

These challenges are evidenced in recent research where investigators noted: "Because there is no suitable antibody to detect endogenous DPY19L3, we could not confirm the KO of DPY19L3 by western blot" .

What alternative methods can researchers use to detect and study DPY19L3 when antibodies are unavailable?

In the absence of reliable antibodies, researchers can employ several alternative approaches to detect and characterize DPY19L3:

• Genetic analysis:

  • Sequence analysis to confirm genetic alterations, as demonstrated in DPY19L3-knockout studies using CRISPR/Cas9

• Epitope tagging:

  • Fusion of DPY19L3 with detectable tags like Gaussia luciferase (Gluc), which allows for protein detection via anti-Gluc antibodies

• Functional assays:

  • Enzymatic activity measurements using mass spectrometry to detect C-mannosylation of known substrates like R-spondin1

  • Phenotypic assays such as vasculogenic mimicry formation on Matrigel and cell proliferation assays that serve as indirect indicators of DPY19L3 presence and function

How can researchers validate DPY19L3 knockout models in the absence of specific antibodies?

Validating DPY19L3 knockout models requires a multi-faceted approach when specific antibodies are unavailable:

• Genomic verification:

  • Sequence analysis to confirm frameshift mutations that generate premature stop codons within the DPY19L3 gene

  • PCR amplification and sequencing of the targeted genomic region to verify the desired mutations

• Transcriptional analysis:

• Functional validation:

  • Vasculogenic mimicry assays on Matrigel, which shows significant inhibition in DPY19L3-KO cell lines compared to mock controls

  • Proliferation assays demonstrating reduced growth in knockout lines

  • Rescue experiments whereby reintroduction of wild-type DPY19L3 (but not inactive isoform2) restores VM formation and cell growth capabilities

What are the best experimental models for studying DPY19L3 function?

Based on current research, several experimental models have proven valuable for investigating DPY19L3 function:

• Cell line models:

  • HT1080 human fibrosarcoma cells, which express DPY19L3 endogenously and demonstrate VM formation capability

  • MDA-MB-231 human breast cancer cells, which also exhibit DPY19L3-dependent VM capacity

  • Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, useful for studying glycosylation-related functions

• Genetic modification approaches:

  • Lentiviral systems for:

    • Overexpression of wild-type or mutant DPY19L3 constructs

    • shRNA-mediated knockdown of DPY19L3

How can researchers design epitopes for DPY19L3 antibody development considering its transmembrane topology?

Designing effective epitopes for DPY19L3 antibody development requires careful consideration of its complex transmembrane topology:

• Topological considerations:

  • Target the C-terminal luminal region, which is critical for enzymatic activity and absent in inactive isoform2

  • Avoid the 11 transmembrane regions and two re-entrant loops identified in topological analysis

  • Consider accessible regions in the cytoplasmic N-terminal domain

• Epitope design strategy:

  • Utilize the experimentally verified topology which shows DPY19L3 comprises 11 transmembrane regions with N-terminal facing cytoplasm and C-terminal in ER lumen

  • Select peptide sequences from exposed loops, particularly avoiding N-glycosylation sites at Asn 118 and Asn 704

  • Avoid sequences with high homology to other DPY19 family members to ensure specificity

• Post-translational modification awareness:

  • Account for the N-glycosylation at Asn 118 and Asn 704 when designing epitopes, as these modifications may alter antibody accessibility

  • Consider that unmodified synthetic peptides used for immunization may generate antibodies that poorly recognize the natively glycosylated protein

What methodological approaches are optimal for measuring DPY19L3 C-mannosyltransferase activity?

The assessment of DPY19L3 C-mannosyltransferase activity requires sophisticated methodological approaches:

• Mass spectrometry-based detection:

  • Analyze C-mannosylation of known DPY19L3 substrates such as R-spondin1 at tryptophan residues W153 and W156

  • Compare wild-type, knockout, and rescue experimental conditions to validate specificity

• Substrate selection considerations:

  • Use R-spondin1 as a validated substrate

  • Consider thrombospondin type 1 repeat (TSR) containing proteins as potential substrates for analysis

• Comparative analysis with DPY19L3 variants:

  • Include isoform2 (lacking C-terminal luminal region) as a negative control, as it lacks C-mannosyltransferase activity

  • Utilize N-glycosylation-defective mutants to assess the relationship between glycosylation and enzymatic function

• Functional correlation assays:

  • Measure downstream effects such as VM formation capabilities, which correlate with DPY19L3 C-mannosyltransferase activity

  • Assess activation of signaling pathways potentially affected by C-mannosylation, such as Akt phosphorylation levels

How can researchers distinguish between different DPY19L3 isoforms in experimental settings?

Distinguishing between DPY19L3 isoforms presents challenges but can be accomplished through several techniques:

• Isoform-specific PCR:

  • Design primers that span the junction regions specific to each isoform

  • For isoform2 (lacking C-terminal region), primers should target the unique splicing junction

• Expression constructs with differential tagging:

  • Generate constructs expressing different isoforms with distinct tags

  • Example from literature: pCI-neo-DPY19L3-Gluc for wild-type and pCI-neo-DPY19L3(isoform2)-Gluc for isoform2

• Functional discrimination:

  • Use C-mannosyltransferase activity as a discriminating feature, as isoform2 lacks enzymatic activity

  • Compare VM formation capability between isoforms, as only wild-type DPY19L3 promotes this process

• Size discrimination:

  • Utilize SDS-PAGE mobility differences due to structural variations between isoforms

  • Consider the impact of differential N-glycosylation patterns on apparent molecular weight

What experimental approaches can assess the role of N-glycosylation in DPY19L3 function?

Investigating the relationship between N-glycosylation and DPY19L3 function can be approached methodically:

• Site-directed mutagenesis:

  • Generate point mutations at confirmed N-glycosylation sites (Asn 118 and Asn 704)

  • Create single and double mutants to assess individual and combined contributions of glycosylation sites

• Glycosylation inhibition:

  • Treat cells with tunicamycin to broadly inhibit N-glycosylation

  • Use PNGase F treatment of purified protein to remove N-glycans enzymatically

• Functional comparative analysis:

  • Assess C-mannosyltransferase activity of wild-type vs. N-glycosylation-defective mutants

  • Evaluate protein stability, localization, and VM formation capability of glycosylation variants

• Structural impact assessment:

  • Investigate how N-glycosylation affects protein folding and membrane integration

  • Consider the relationship between glycosylation status and the critical C-terminal region function

Interestingly, previous research has shown that while N-glycosylations occur at specific sites in DPY19L3, they "do not have any roles for its enzymatic activity" , suggesting their importance may lie in other aspects of protein function or regulation.

What methodological considerations are important for studying DPY19L3's role in cancer progression?

Investigating DPY19L3's role in cancer progression requires careful methodological considerations:

• Model selection and validation:

  • Utilize multiple cancer cell lines to ensure broad applicability:

    • HT1080 (human fibrosarcoma cells)

    • MDA-MB-231 (human breast cancer cells)

  • Validate findings in primary patient-derived cancer cells where possible

• Functional assays for cancer-relevant phenotypes:

  • Vasculogenic mimicry formation on Matrigel (standardized protocol with 24-hour assessment)

  • Proliferation assays with standardized seeding (1.0 × 10³ cells/well) and time points (0h, 72h)

  • Migration and invasion assays to assess metastatic potential

• Pathway analysis:

  • Western blot analysis of Akt and phosphorylated Akt levels as demonstrated in previous research

  • Investigation of Wnt signaling effects given DPY19L3's role in R-spondin1 C-mannosylation

• In vivo validation approaches:

  • Consider xenograft models with DPY19L3-knockout or overexpressing cancer cells

  • Assess tumor growth, angiogenesis, and metastatic capability