Glutamate carboxypeptidase II (GCPII) is usually a membrane-bound binuclear zinc metallopeptidase with the highest expression levels found in the nervous and prostatic tissue. GCPII internalization and recycling . GCPII is usually internalized in a constitutive manner, yet the internalization rate is usually increased by the binding of GCPII-specific antibodies to the extracellular domain name of the protein . These findings are being exploited for the development of therapeutic approaches to target the delivery of toxins, drugs, and short-range isotopes to the interior of GCPII-expressing cells. The bulk of the protein is usually oriented to the extracellular milieu, where it can take action on its natural substrates (Fig. 1; observe Chapter 3.3). The extracellular portion of GCPII homodimerizes and the dimerization is usually believed to be required for GCPII hydrolytic activity , even though the active site in each subunit is usually structurally impartial . GCPII is also greatly N- and O-glycosylated (glycans can account for up to 25% of the total molecular weight of the protein); you will find ten N-glycosylation sites predicted within the primary sequence of human GCPII and the N-glycosylation is usually indispensable for GCPII enzymatic activity and stability [8C12]. Furthermore, glycosylation of the protein is usually implicated in apical sorting, proteolytic resistance and its association with lipid rafts [13,14]. Open in a separate windows Fig. (1) Homodimer of human GCPII (crystal structure) tethered to the biological membrane. One monomer shown in semitransparent surface representation with individual domains of the extracellular part colored green (protease domain name; amino acids 57 C 116 and 352 C 590), blue (apical domain name; amino acids 117 C 351), and yellow (C-terminal; amino acids 591 C 750); the second monomer is usually colored gray. N-linked sugar moieties are colored cyan, and the active-site Zn2+ ions are shown as reddish spheres. Left panel C residing at the plasma membrane of astrocytes /schwann cells, GCPII catabolizes NAAG, the most prevalent peptidic neurotransmitter in the mammalian nervous system. N-acetylaspartate and glutamate, the reaction products, are selectively transported into glial cells, metabolized and reused for NAAG synthesis in neurons. Right panelC GCPII (or folate hydrolase) at the plasma membrane of enterocytes in the proximal jejunum sequentially hydrolyzes the C-terminal -glutamate tail of dietary folates, finally leaving folate-monoglutamate, which can be then transported transcellularly into the blood stream. 2.2. Tertiary Structure The 3-dimensional structure of the human GCPII ectodomain was solved by two groups independently [7, 15]. The overall fold closely resembles the structure of the transferrin receptor . The extracellular a part of GCPII consists of three unique domains spanning amino acids 57C116 and 352C590 (the protease domain name), 117C351 (the apical 17560-51-9 supplier domain name), and 591C750 (the C-terminal or dimerization domain name). Synergetic action of all three 17560-51-9 supplier domains is required for productive substrate binding and processing, as several residues from each domain name contribute to the architecture of the GCPII substrate binding cavity and are 17560-51-9 supplier involved in ligand acknowledgement . The GCPII substrate binding cavity is usually divided by the active site (featuring two zinc ions) into two halves, designated the S1 pocket and the S1 pocket, respectively. The binuclear zinc active site, with the two zinc ions coordinated by the side chains of His377, Asp387, Glu425, Asp453, and His553, is usually indispensable for the GCPII hydrolytic activity [17,18]. It is also exploited for the design of high-affinity inhibitors as every high-affinity GCPII inhibitor includes Rabbit Polyclonal to SIX3 a zinc-binding group in its structure. Amino acid residues shaping S1 and S1 pouches dictate GCPII preferences towards physicochemical characteristics of cognate substrates and small-molecule inhibitors. The 17560-51-9 supplier S1 pocket, also termed a pharmacophore pocket, is usually optimized for binding of glutamate and glutamate-like moieties [19C21]. Not surprisingly then, both known natural GCPII substrates (NAAG and folyl-poly–glutamates) feature glutamate as the C-terminal residue. Additionally, the majority of GCPII-specific inhibitors are derived from glutamate (or NAAG) scaffolds to take the advantage of S1 pocket affinity towards.