ANPEP - alanyl aminopeptidase membrane |Elisa - Clia - Antibody - Protein

Background

ANPEP, also known as alanyl aminopeptidase or aminopeptidase N (APN), is a key membrane-bound enzyme that plays a significant role in the hydrolysis of peptides. The gene ANPEP encodes this enzyme, which is expressed predominantly on the surface of epithelial cells in the small intestine, kidney, and other tissues, and also in certain immune cells such as monocytes, macrophages, and granulocytes. ANPEP is classified as a zinc metalloprotease, which means that its catalytic activity depends on a zinc ion in its active site.

In the small intestine, ANPEP facilitates the final stages of protein digestion by cleaving neutral amino acids from the N-terminus of oligopeptides. Beyond its digestive role, ANPEP also participates in various physiological processes, including antigen processing, angiogenesis, immune response regulation, and the inflammatory response. Due to its broad distribution and diverse roles, ANPEP is involved in a wide range of biological functions, from nutrient absorption to immune cell function, and it has been implicated in several clinical conditions, including cancer, viral infections, and inflammatory diseases.

Protein Structure

The structure of ANPEP (aminopeptidase N) is critical to understanding its diverse functions. ANPEP is a large type II transmembrane glycoprotein consisting of approximately 967 amino acids in humans. The protein's structure can be divided into several distinct regions that contribute to its catalytic activity, cellular localization, and interactions with other proteins.

N-terminal Cytoplasmic Domain:

• ANPEP begins with a short N-terminal cytoplasmic tail that anchors the protein to the inner side of the plasma membrane. This domain is relatively short and does not play a significant role in the enzyme’s catalytic activity, but it is important for the proper localization of the protein at the cell surface.

Transmembrane Domain:

• Following the cytoplasmic domain, ANPEP possesses a single transmembrane helix, which spans the cell membrane and anchors the protein in place. This region allows ANPEP to act as a membrane-bound enzyme, meaning it can hydrolyze substrates at the extracellular surface while remaining tethered to the cell.

Extracellular Domain:

• The majority of ANPEP's structure consists of the extracellular domain, which extends outward from the cell membrane. This domain is composed of several key regions:
• Catalytic domain: The active site of ANPEP is located within its extracellular domain. The enzyme is classified as a zinc-dependent metalloprotease, meaning its activity requires a zinc ion coordinated at the catalytic site. The zinc ion is essential for the hydrolysis of peptide bonds, and it is coordinated by residues within the active site, including a critical HEXXH motif (common to metalloproteases), where two histidine residues bind to the zinc ion. The third zinc ligand is typically a glutamate residue, which also plays a role in the catalytic mechanism. The enzyme functions by cleaving N-terminal neutral amino acids from peptides, which is crucial for the breakdown of dietary proteins and various physiological processes.
• Substrate recognition site: ANPEP's substrate specificity is due to the arrangement of residues within its active site, which favor the binding of peptides that contain neutral amino acids at their N-terminus. The enzyme's conformation allows it to selectively interact with peptides and process them for further degradation or absorption.
• Glycosylation sites: ANPEP is heavily glycosylated, with multiple N-linked glycosylation sites in its extracellular domain. Glycosylation is important for protein stability, proper folding, and interactions with other proteins or substrates.

The three-dimensional structure of ANPEP has been solved using X-ray crystallography, revealing a structure consisting of multiple α-helices and β-sheets, with a central pocket for substrate binding and catalysis. The extracellular domain is highly flexible, allowing it to accommodate various peptide substrates, and the enzyme is thought to function as a dimer, which may enhance its catalytic efficiency or modulate its interactions with other molecules at the cell surface.

Classification and Subtypes

ANPEP is classified under the family of zinc-dependent metalloproteases. It belongs to the M1 family of metalloproteases, which are also known as gluzincins due to the conserved glutamate residue that plays a role in coordinating the zinc ion in the active site. Within this family, ANPEP is one of the most prominent members due to its wide distribution and variety of functions.

ANPEP does not have distinct subtypes, but its expression is regulated in a tissue-specific manner. It is highly expressed in:

• Epithelial cells of the small intestine, where it functions in protein digestion.
• Renal epithelial cells, where it plays a role in reabsorption of peptides in the kidney.
• Myeloid cells, such as macrophages and granulocytes, where it is involved in immune responses.

There are several related enzymes in the M1 family, such as ERAP1 (endoplasmic reticulum aminopeptidase 1) and ERAP2, which play roles in antigen processing in immune cells. However, these are distinct from ANPEP and have specialized roles in the immune system.

Function and Biological Significance

ANPEP plays a critical role in multiple physiological processes:

Peptide Digestion and Absorption:

• The primary function of ANPEP in the small intestine is to facilitate the final stages of protein digestion. It cleaves neutral amino acids from the N-terminus of small peptides that have already been broken down by other proteases, such as trypsin and chymotrypsin. This final step is crucial for allowing dipeptides, tripeptides, and amino acids to be absorbed by enterocytes in the intestinal lining.

Immune Function:

• ANPEP is expressed on the surface of monocytes, macrophages, granulocytes, and certain dendritic cells, where it plays a role in the immune system. It is involved in antigen processing, where it trims peptides for presentation on MHC class I molecules to T cells, a crucial step in the initiation of immune responses.
• Additionally, ANPEP has been implicated in inflammation regulation, where it modulates the activity of bioactive peptides that participate in inflammatory processes.

Viral Infection:

• ANPEP acts as a receptor for certain coronaviruses, including the porcine epidemic diarrhea virus (PEDV) and the human coronavirus 229E (HCoV-229E). These viruses use ANPEP as an entry point into host cells, which underscores its role in viral pathogenesis and highlights its importance as a potential target for therapeutic interventions in viral diseases.

Angiogenesis:

• ANPEP is involved in angiogenesis, the formation of new blood vessels. It has been shown to be upregulated in the endothelial cells of tumor vasculature, where it promotes angiogenesis. This makes ANPEP a potential therapeutic target in cancer, particularly in strategies aiming to inhibit the blood supply to tumors.

Renal Function:

• In the kidneys, ANPEP is expressed on proximal tubule cells, where it participates in the reabsorption and degradation of filtered peptides. It helps in maintaining the balance of peptides in the bloodstream and contributes to the overall function of the renal system.

Clinical Issues

ANPEP is implicated in several diseases, ranging from viral infections to cancer:

Cancer:

• ANPEP is overexpressed in several types of cancer, including colorectal cancer, renal cell carcinoma, and lung cancer. Its role in promoting angiogenesis makes it a key player in tumor growth and metastasis. Targeting ANPEP with inhibitors or antibodies is being explored as a strategy to reduce tumor vascularization and starve cancer cells of nutrients.

Inflammatory Diseases:

• Due to its involvement in immune regulation and inflammation, dysregulation of ANPEP activity can contribute to chronic inflammatory conditions, such as rheumatoid arthritis and inflammatory bowel disease (IBD). In these conditions, ANPEP may modulate the levels of inflammatory peptides, exacerbating or alleviating inflammation.

Viral Infections:

• As a receptor for coronaviruses, including the common cold-causing HCoV-229E, ANPEP facilitates viral entry into host cells. While it is not directly involved in the current COVID-19 pandemic (which involves SARS-CoV-2 and the ACE2 receptor), its role in other coronavirus infections makes it an important target for antiviral drug development.

Kidney Disorders:

• Given its role in peptide reabsorption in the kidneys, mutations or dysregulation of ANPEP may be implicated in renal diseases. However, more research is needed to fully elucidate its role in these conditions.

Summary

ANPEP (Alanyl Aminopeptidase N) is a critical membrane-bound enzyme involved in various physiological processes, including protein digestion, immune regulation, and angiogenesis. Its structure, featuring a zinc-dependent active site, allows it to cleave neutral amino acids from the N-terminus of peptides, facilitating peptide degradation and absorption. Beyond its role in digestion, ANPEP is crucial for immune functions such as antigen processing and has been implicated in inflammatory responses, viral infections, and tumor angiogenesis. Clinically, ANPEP is a potential target for therapies against cancer, viral diseases, and chronic inflammatory conditions due to its multifaceted roles in health and disease.