DPEP1 Antibody

What is DPEP1 and what are its key physiological functions?

DPEP1 (also known as MDP, RDP, renal dipeptidase, or microsomal dipeptidase) is a zinc-dependent membrane-bound glycoprotein belonging to the peptidase M19 family. Its principal functions include:

• Hydrolysis of various dipeptides, particularly those containing hydrophobic amino acids

• Conversion of leukotriene D4 to leukotriene E4, regulating inflammatory processes

• Hydrolysis of cystinyl-bis-glycine formed during glutathione degradation

• Beta-lactamase activity capable of hydrolyzing beta-lactam antibiotics like imipenem

• Function as an adhesion receptor for neutrophil recruitment from bloodstream into inflamed tissues (lungs, liver, kidneys)

These diverse activities position DPEP1 at the intersection of inflammation, metabolism, and immunity, making it a significant target for research in multiple disease contexts.

What is the tissue distribution and subcellular localization of DPEP1?

DPEP1 exhibits a highly specific tissue distribution pattern:

Primary expression locations:

• Kidney proximal tubular epithelial cells (highest expression)

• Peritubular capillaries in kidney (expression increases during injury)

• Brush border of proximal tubular cells in mouse kidneys

Subcellular localization:

• Primarily a membrane-bound protein via GPI anchor

• Concentrated in the brush border membrane of proximal tubules

• Also present in peritubular endothelial cells

Using immunohistochemistry, DPEP1 is visualized primarily in the proximal tubules of normal human kidney . Following inflammatory stimuli like ischemia-reperfusion injury (IRI) or lipopolysaccharide (LPS) administration, DPEP1 expression increases substantially in both tubular and vascular compartments .

How is DPEP1 regulated during disease states and inflammation?

DPEP1 expression is dynamically regulated in response to various pathological conditions:

Inflammatory regulation:

• Protein expression increases in kidney homogenates within 8 hours of ischemia-reperfusion injury (IRI)

• Systemic LPS administration similarly increases DPEP1 expression

• Both IRI and LPS induce post-translational modifications that increase DPEP1's molecular mass

Cancer-related regulation:

• Significantly upregulated in colorectal cancer compared to normal mucosa (P<0.0001)

• Shows striking upregulation during transition from low-grade to high-grade intraepithelial neoplasia (IEN)

• Expression correlates strongly with tumor stage (P<0.0001) and localization (P<0.0001)

• Interestingly, DPEP1 is downregulated in other cancer types (Wilms' tumors, breast lobular carcinomas, pancreatic adenocarcinomas)

Kidney transplantation:

• Increased expression in peritubular capillaries of injured kidneys from transplant patients experiencing IRI

This differential regulation suggests context-specific roles for DPEP1 in different disease states.

What are the critical considerations when selecting a DPEP1 antibody?

Selecting an appropriate DPEP1 antibody requires careful consideration of several factors:

Table 1: Key Criteria for DPEP1 Antibody Selection

Most commercial DPEP1 antibodies are rabbit polyclonals, though mouse monoclonals are also available. For critical experiments, testing multiple antibodies targeting different epitopes is recommended to confirm specificity and reproducibility.

What validation approaches are essential for confirming DPEP1 antibody specificity?

A multi-faceted approach to DPEP1 antibody validation is strongly recommended:

• Molecular weight verification:

  • DPEP1 should appear at approximately 45-46 kDa on Western blots

  • Be aware that a non-specific band at ~70-75 kDa may appear in tissue lysates but not cell lines

• Knockout/knockdown validation:

  • Use DPEP1 knockout or knockdown samples as negative controls

  • Several publications have validated antibodies using this approach

• Multiple antibody comparison:

  • Use antibodies targeting different DPEP1 epitopes

  • Consistent results across different antibodies increase confidence in specificity

• Tissue distribution analysis:

  • Verify strong staining in kidney proximal tubules (known high expression)

  • Compare expression patterns with published literature

• Peptide competition assay:

  • Pre-incubation with immunizing peptide should abolish specific signal

  • Non-specific binding will typically remain

For researchers studying DPEP1 in disease contexts, comparing normal vs. pathological samples provides additional validation, as DPEP1 shows characteristic expression changes in conditions like colorectal cancer and kidney inflammation .

Which positive controls are most reliable for DPEP1 antibody validation?

Selecting appropriate positive controls is critical for DPEP1 antibody validation:

Tissue controls:

• Kidney tissue (particularly proximal tubules) – primary site of physiological expression

• Colorectal cancer tissue – shows upregulated expression compared to normal mucosa

• Liver tissue – also expresses detectable DPEP1

Cell line controls:

• HepG2 cells – validated as DPEP1-positive for Western blotting

• HEK-293 cells – show detectable DPEP1 expression

• Selected colon cancer cell lines – 5 out of 7 tested lines showed enriched expression

Recombinant protein:

• Purified recombinant DPEP1 for absolute specificity confirmation

• Particularly useful for antibodies raised against recombinant immunogens

Recommendation for critical applications:
Include both tissue and cell line positive controls when possible, as expression levels and patterns may differ. Kidney tissue sections should show characteristic brush border staining in proximal tubules, providing a distinctive pattern that helps confirm antibody specificity.

What are the optimized protocols for DPEP1 detection by Western blotting?

Successful DPEP1 Western blotting requires attention to several key protocol elements:

Sample preparation:

• For tissue samples: Homogenize and lyse in buffer containing 0.1% SDS, 1% Nonidet P-40, 0.5% sodium desoxycholate, 1 mM EGTA and 1 mM sodium vanadate

• Include fresh protease inhibitors to prevent degradation

• For cell lines: Standard RIPA or NP-40 based lysis buffers generally work well

Protocol parameters:

• Protein loading: 20-50 μg total protein per lane

• Gel percentage: 10-12% SDS-PAGE gels provide good resolution

• Transfer: Semi-dry or wet transfer (add 0.1% SDS to transfer buffer to improve transfer of membrane proteins)

• Blocking: 5% non-fat milk or BSA in TBST (1 hour at room temperature)

• Primary antibody: 1:1000-1:5000 dilution in blocking buffer (overnight at 4°C)

• Secondary antibody: HRP-conjugated, matched to host species (1:5000-1:10000)

• Detection: Enhanced chemiluminescence systems work well for DPEP1

Expected results:

• A specific band at 45-46 kDa corresponding to DPEP1

• Potential non-specific band at ~70-75 kDa in tissue lysates

• Possible slight increases in molecular mass following inflammatory stimuli due to post-translational modifications

Quantification:

• Normalize to appropriate loading controls (β-actin commonly used)

• For comparing expression across conditions, include multiple biological replicates

What are the key considerations for immunohistochemical detection of DPEP1?

Optimized immunohistochemistry protocols for DPEP1 include:

Tissue preparation:

• Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used

• Section thickness: 4-5 μm for optimal staining and resolution

• For tissue microarrays (TMAs), multiple cores per sample (2-3) improve reliability

Staining protocol:

• Deparaffinization and rehydration: Standard xylene/ethanol series

• Antigen retrieval: Some DPEP1 antibodies work without retrieval ; for others, test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

• Blocking: 5-10% normal serum or commercial blocking reagent

• Primary antibody: Typically 1:500 dilution, incubate 1-2 hours at room temperature or overnight at 4°C

• Detection system: Both biotin-based and polymer-based systems have been successfully used

• Counterstain: Hematoxylin for nuclear visualization

Evaluation guidelines:

• Staining intensity: Evaluate as weak, moderate, or strong

• Positivity threshold: ≥10% of cells showing DPEP1 staining is commonly used

• Localization patterns:

  • Normal kidney: Strong brush border staining in proximal tubules

  • CRC tissue: Cytoplasmic and membranous staining, intensity correlates with grade

Special considerations:

• Include both normal and pathological tissues on the same slide when possible

• For colorectal samples, examine transition areas between normal and neoplastic tissue to observe gradients in DPEP1 expression

How can I resolve commonly encountered problems with DPEP1 antibodies?

Researchers frequently encounter several challenges when working with DPEP1 antibodies:

Table 2: Common DPEP1 Antibody Issues and Solutions

When troubleshooting, always include appropriate positive controls and consider testing multiple antibodies targeting different regions of DPEP1 to determine whether the issue is antibody-specific or related to DPEP1 biology in your experimental system.

How can DPEP1 antibodies be used to investigate its role in inflammation and neutrophil recruitment?

DPEP1's emerging role in leukocyte adhesion and inflammatory processes can be investigated using several antibody-based approaches:

Cellular localization studies:

• Dual immunofluorescence staining with endothelial markers (CD31) and DPEP1 to visualize expression in inflamed vessels

• Flow cytometry to quantify DPEP1 expression on endothelial cells and epithelial cells before and after inflammatory stimuli

• Live-cell imaging using fluorescently labeled antibodies to track DPEP1 redistribution during inflammation

Functional assays:

• Neutrophil adhesion assays using anti-DPEP1 blocking antibodies to confirm functional role

• Immunoprecipitation to identify DPEP1-interacting partners in the neutrophil adhesion cascade

• ChIP assays to identify transcription factors regulating DPEP1 expression during inflammatory responses

In vivo approaches:

• Intravital microscopy with fluorescently labeled leukocytes to visualize recruitment in DPEP1 knockout vs. wild-type animals

• Tissue immunostaining to quantify neutrophil infiltration in relation to DPEP1 expression

• Therapeutic studies using DPEP1-targeting peptides (like LSALT) in inflammatory disease models

Mechanistic investigations:

• Phospho-specific antibodies to identify activation-dependent modifications of DPEP1

• Antibodies recognizing specific post-translational modifications that correlate with DPEP1's adhesion function

• Epitope mapping to identify regions critical for neutrophil binding

These approaches collectively can elucidate the mechanisms by which DPEP1 transitions from an enzymatic protein to an adhesion receptor during inflammation.

What methods are most effective for studying DPEP1 in cancer progression?

DPEP1's significant association with colorectal cancer progression can be investigated using these approaches:

Expression analysis across cancer stages:

• Immunohistochemical evaluation of DPEP1 across the adenoma-carcinoma sequence

• Special focus on the transition from low-grade to high-grade intraepithelial neoplasia (IEN), where DPEP1 shows striking upregulation

• Quantitative analysis using digital pathology for precise correlation with histopathological features

Prognostic significance assessment:

• Tissue microarray analysis of large patient cohorts with follow-up data

• Correlation of DPEP1 expression with patient survival and clinical parameters

• Multivariate analysis incorporating DPEP1 expression with established prognostic markers

Molecular mechanisms:

• Co-immunoprecipitation to identify cancer-specific DPEP1 interacting partners

• Chromatin immunoprecipitation to identify transcriptional regulators of DPEP1 in cancer

• Antibody-based proteomics to identify post-translational modifications specific to cancer-associated DPEP1

Functional studies:

• Knockdown/knockout studies combined with antibody-based detection of phenotypic changes

• Analysis of cancer-related signaling pathways affected by DPEP1 modulation

• Therapeutic targeting studies using antibodies or peptides against DPEP1

Methodological considerations:

• Use standardized scoring systems for IHC (weak/moderate/strong intensity with ≥10% positivity threshold)

• Include ROC curve analysis to determine optimal cutoff values for prognostication

• Compare protein expression (antibody-based detection) with mRNA expression for comprehensive analysis

How can DPEP1 antibodies be applied to study viral interactions with DPEP1?

The recent discovery of DPEP1 as a functional receptor for coronavirus PHEV opens new research avenues using antibody-based approaches:

Receptor-virus interaction studies:

• Co-immunoprecipitation with anti-DPEP1 antibodies to pull down viral spike proteins

• Proximity ligation assays to visualize DPEP1-spike protein interactions in situ

• Competition assays using anti-DPEP1 antibodies to block viral binding

Critical binding sites identification:

• Epitope-specific antibodies to map regions of DPEP1 involved in virus binding

• Site-directed mutagenesis of key residues (e.g., E351) followed by antibody-based detection of binding

• Structural studies of DPEP1-spike complexes using antibody fragments for crystallization

Infection inhibition strategies:

• Development of neutralizing antibodies targeting the DPEP1 virus-binding domain

• Testing therapeutic efficacy of existing anti-DPEP1 antibodies in viral infection models

• Combination approaches targeting both DPEP1 and viral proteins

Cellular expression and trafficking:

• Flow cytometry to quantify DPEP1 expression levels on susceptible cell types

• Immunofluorescence to track DPEP1 redistribution following viral exposure

• Live-cell imaging to visualize virus-induced changes in DPEP1 localization

These approaches can be adapted to study potential roles of DPEP1 in other viral infections, particularly within the coronavirus family, which may utilize similar receptor mechanisms.

What techniques can distinguish enzymatic versus adhesion functions of DPEP1?

DPEP1 has dual functions as both an enzyme and an adhesion receptor, requiring specialized approaches to differentiate these roles:

Functional domain mapping:

• Use domain-specific antibodies to block either enzymatic activity or adhesion function

• Immunoprecipitation with conformation-specific antibodies that recognize active vs. inactive enzyme states

• Correlate post-translational modifications with specific functions using modification-specific antibodies

Activity-based assays:

• Measure enzymatic activity (leukotriene conversion, dipeptide hydrolysis) in parallel with adhesion assays

• Use LSALT peptide (blocks adhesion but not enzymatic function) as a tool to differentiate functions

• Combine with antibody detection to correlate protein levels with specific activities

Site-directed functional studies:

• Create point mutations that selectively impair enzymatic function or adhesion

• Use antibodies to confirm expression levels while measuring functional outcomes

• Identify residues (like E351) critical for specific functions

Temporal and spatial regulation:

• Immunofluorescence to visualize DPEP1 redistribution during activation of adhesion function

• Phospho-specific antibodies to identify activation-dependent modifications

• Flow cytometry to quantify surface expression changes correlating with functional transitions

Methodological considerations:

• Ensure antibodies used don't interfere with the function being measured

• Include appropriate functional controls (e.g., cilastatin as enzymatic inhibitor)

• Consider the impact of membrane microenvironment on both functions when designing assays

How are DPEP1 antibodies being used in biomarker development?

DPEP1's potential as a biomarker is being explored through several antibody-based approaches:

Colorectal cancer screening and prognosis:

• IHC-based evaluation of DPEP1 in colorectal neoplasia, with particularly high expression in high-grade IEN

• ROC analysis demonstrating DPEP1's prognostic value (ROC-AUC of 0.9230)

• Development of standardized scoring systems for clinical implementation

Diagnostic applications:

• Detection of DPEP1 in circulating tumor cells as a biomarker for metastatic CRC

• Assessment in precancerous lesions to identify high-risk patients

• Combination with other markers to improve diagnostic accuracy

Inflammatory kidney disease:

• Monitoring DPEP1 expression in kidney biopsies as a marker of inflammatory damage

• Potential predictive marker for acute kidney injury following ischemia-reperfusion

• Correlating expression patterns with treatment response

Methodological advances:

• Development of high-sensitivity immunoassays for DPEP1 detection in biological fluids

• Multiplex IHC approaches combining DPEP1 with other diagnostic markers

• Digital pathology algorithms for standardized DPEP1 quantification

Clinical validation stages:

• Technical validation of antibody specificity across multiple cohorts

• Clinical validation in prospectively collected samples

• Multicenter studies to establish standardized protocols

• Assessment of impact on clinical decision-making and patient outcomes

What are the technical challenges in studying post-translational modifications of DPEP1?

Investigating DPEP1's post-translational modifications presents several technical challenges:

Identification challenges:

• DPEP1 undergoes multiple modifications including glycosylation and possibly phosphorylation

• Inflammatory stimuli induce modifications that slightly increase molecular mass

• These modifications may be tissue-specific and context-dependent

Methodological approaches:

• Phospho-specific antibodies:

  • Development of antibodies recognizing specific phosphorylation sites

  • Use of phosphatase treatments as controls to confirm specificity

• Glycosylation analysis:

  • Enzymatic deglycosylation followed by Western blotting to reveal mobility shifts

  • Lectin affinity approaches combined with anti-DPEP1 antibodies

  • Mass spectrometry to identify specific glycan structures

• Site-specific mutation studies:

  • Create mutations at putative modification sites

  • Use antibodies to detect expression and localization changes

• Advanced microscopy:

  • Super-resolution microscopy to visualize DPEP1 clustering dependent on modifications

  • FRET-based approaches to detect modification-dependent conformational changes

Key research questions:

• Which modifications switch DPEP1 from enzymatic to adhesion function?

• How do inflammatory stimuli trigger specific modifications?

• Which enzymes mediate these critical post-translational changes?

Addressing these challenges requires integrating antibody-based detection with advanced proteomic and imaging techniques for comprehensive characterization of DPEP1 modifications.