XPNPEP1 Antibody

What is XPNPEP1 and what is its biochemical function?

XPNPEP1 (X-Prolyl Aminopeptidase 1) is a cytosolic metalloaminopeptidase that catalyzes the removal of penultimate prolyl residues from the N-termini of peptides, such as Arg-Pro-Pro. It plays a significant role in the degradation of bradykinin and related peptides . The protein possesses a zinc-binding motif essential for its enzymatic activity. XPNPEP1 is also known by several alternative names including XPNPEPL, XPNPEPL1, Xaa-Pro aminopeptidase 1, aminoacylproline aminopeptidase, cytosolic aminopeptidase P, soluble aminopeptidase P, and sAmp .

Unlike its membrane-bound counterpart XPNPEP2 (which is GPI-anchored to the plasma membrane), XPNPEP1 functions as a soluble, cytosolic enzyme . Both enzymes are metalloproteases that utilize manganese as a cofactor for their catalytic activity .

What is the structural classification of XPNPEP1 and its evolutionary conservation?

XPNPEP1 belongs to the M24 family of metalloproteases, which includes methionine aminopeptidases, X-Pro dipeptidase, aminopeptidase P2, aminopeptidase P homolog, proliferation-associated protein 1, and the suppressor of Ty homolog/chromatin-specific transcription elongation factor large subunit .

The protein demonstrates remarkable evolutionary conservation across species. Human XPNPEP1 shares 99% amino acid sequence identity with canine, 97% with bovine, 95% with mouse/rat, 74% with Xenopus, and 73% with zebrafish homologs . This high degree of conservation suggests fundamental biological importance across vertebrate species.

What experimental applications are validated for XPNPEP1 antibodies?

XPNPEP1 antibodies have been validated for multiple applications including immunohistochemistry on paraffin-embedded tissues (IHC-P), Western blotting (WB), immunocytochemistry/immunofluorescence (ICC/IF), and enzyme-linked immunosorbent assay (ELISA) . When selecting an antibody, researchers should consider the specific experimental requirements including tissue/cell type, fixation methods, and detection systems. For instance, monoclonal antibodies like OTI1E3 offer high specificity and reproducibility across multiple applications, while polyclonal antibodies may provide enhanced sensitivity through epitope diversity .

How does XPNPEP1 expression differ between normal and malignant tissues, particularly in renal cell carcinoma?

Research on clear cell renal cell carcinoma (ccRCC) in von Hippel-Lindau (VHL) patients has revealed intriguing differential expression patterns of XPNPEP1 and XPNPEP2. Immunohistochemical analysis demonstrates that while both proteases are abundantly expressed in the proximal tubuli of normal kidney tissue, ccRCC exhibits significantly elevated levels of XPNPEP1 (p<0.05) alongside significantly decreased levels of XPNPEP2 (p<0.01) .

This opposing regulation pattern suggests a potential compensatory mechanism or altered functional requirements in the malignant state. In ccRCC tissue, XPNPEP1 is predominantly expressed by the tumor cells themselves rather than the surrounding stroma or inflammatory cells, as confirmed through careful macrodissection techniques focusing specifically on tumor tissue regions . This represents the first dedicated report of differential regulation of XPNPEP proteases in ccRCC, highlighting a potential role in tumorigenesis or tumor progression.

What are the functional consequences of XPNPEP1 manipulation in cancer cell models?

Functional investigation of XPNPEP1 in cancer has been performed using small-hairpin RNA (shRNA) mediated expression silencing in the 786-O ccRCC cell line, which harbors a mutated VHL gene. Interestingly, XPNPEP1 expression was found to dampen cellular proliferation and migration in these cells .

Western blot analysis of 786-O cells revealed two protein species in the 70-75 kDa range, corresponding to the predicted molecular weights of different XPNPEP1 isoforms. While this may indicate the presence of multiple isoforms, it could also reflect post-translational modifications such as proteolytic truncation or acetylation .

These findings suggest that XPNPEP1 may function as an anti-target in ccRCC, with its elevated expression potentially representing a cellular response to limit tumor aggressiveness. This counterintuitive role highlights the complex nature of protease function in cancer biology and suggests careful consideration when targeting XPNPEP1 in potential therapeutic approaches.

How are XPNPEP1 isoforms characterized and detected in experimental systems?

Four isoforms of XPNPEP1 have been reported with sequence lengths ranging from 599 to 666 amino acids. When detected by immunoblotting, researchers typically observe two protein species in the 70-75 kDa range, corresponding to the predicted molecular weights of these different isoforms .

For optimal detection of XPNPEP1 isoforms via Western blotting, antibodies targeting conserved regions across isoforms, such as the C-terminal epitope LIRETQPISKQH, can recognize multiple variants. The C-terminal antibody (ABIN570995) specifically recognizes isoforms 1 and 2 (NP_065116.2, NP_001161076.1) .

When designing experiments to differentiate between specific isoforms, researchers should consider:

• Using isoform-specific antibodies when available

• Running gradient gels to achieve better separation of closely sized proteins

• Employing isoform-specific PCR primers when analyzing expression at the mRNA level

• Considering mass spectrometry for precise isoform identification

• Using recombinant isoform standards as positive controls

What methodological approaches are recommended for studying XPNPEP1 in tissue samples?

For robust investigation of XPNPEP1 in tissue samples, immunohistochemistry has proven effective, particularly in formalin-fixed paraffin-embedded (FFPE) materials. Quantitative proteomics on FFPE samples has been validated as a reliable approach for studying XPNPEP1 expression patterns .

When performing IHC, proximal tubuli in normal kidney tissue serve as reliable positive controls for XPNPEP1 expression, as these structures consistently demonstrate abundant expression of the protein . For more specific localization studies, immunofluorescence with subcellular markers can help confirm the cytosolic distribution pattern of XPNPEP1, distinguishing it from the membrane-associated XPNPEP2.

Macrodissection techniques are recommended when working with heterogeneous tissue samples to focus specifically on areas of interest and reduce contamination from stromal or inflammatory cells that may confound expression analysis . This approach is particularly valuable when comparing expression patterns between tumor and adjacent normal tissues.

What is known about XPNPEP1's substrate specificity and how can it be assessed experimentally?

XPNPEP1 removes N-terminal amino acids from peptides containing a proline residue at the second position. It specifically cleaves after proline ("P1 proline" in Schechter-Berger nomenclature), removing di- or tri-peptides from protein or peptide N-termini .

While XPNPEP1 and XPNPEP2 are thought to have comparable structural properties and similar substrate specificities, it remains unclear whether they can functionally compensate for each other . Both enzymes have been studied regarding their ability to process bradykinin, but their complete substrate profiles likely include many other biologically relevant peptides.

For experimental assessment of XPNPEP1 enzymatic activity and substrate specificity, researchers can employ:

• Fluorogenic peptide substrates with N-terminal Pro residues

• Mass spectrometry-based approaches to identify cleavage products

• In vitro enzyme assays with purified recombinant XPNPEP1

• Comparative analysis with XPNPEP2 to identify unique versus shared substrates