Maia M. Chernaia

Crystal Structure of Human Pepsinogen A

Human pepsinogen A is the inactive protein precursor of pepsin, an aspartic proteinase found in the stomach. Pepsinogen is synthesized in the chief cells and secreted into the gastric lumen. After pepsinogen has been exposed to the acidic pH of the stomach, a 47 amino acid, N-terminal prosegment is removed by autolytic cleavage to form the active enzyme.

X-ray crystallographic studies of the complex between porcine pepsin and the aspartic proteinase inhibitor PI-3 from the nematode Ascaris suum.

Ascaris suum is an intestinal endoparasitic nematode found in pigs. In order to avoid proteolytic digestion by the host the nematode has developed a large battery of proteinase inhibitors. Among those is the aspartic proteinase inhibitor PI-3. PI-3 consists of a single polypeptide chain containing 149 residues with a molecular weight of 16.4 kD. It has three disulfide bridges. CD and secondary structure predictions indicate that the fold of PI-3 is mixed alpha/beta.1 Until now only a very few larger aspartic proteinase inhibitors have been characterized. Aspartic proteinase inhibitors from the Filarie family of parasites have been reported (Dirofilaria, Brugia and Onchocerca). These inhibitors are more closely related to each other (~60% sequence identity) than PI-3 (less than 20%).2 The inhibitor form Ascaris suum show a broad specificity towards members from the pepsin related family of aspartic proteinases having Ki’s in the range of 0.2—15 nM (porcine and human pepsin and cathepsin E). Cathepsin D however, is not inhibited by PI-3, although it shares up to 45 identity to the above mentioned aspartic proteinases.3 This has important aspects as it might be used to distinguish between cathepsin D and E. The following report describes the progress in the structure determination of the molecular complex between porcine pepsin and PI-3.

The Molecular Structure of Human Progastricsin and its Comparison with that of Porcine Pepsinogen

Mammalian aspartic proteinases are synthesized as inactive precursors or zymogens. Stomach zymogens undergo a conversion to the active enzyme form autocatalytically at pH < 5.0 (1). The human gastric juice has two major groups of aspartic proteinases, the pepsins (EC3.4.23.1) and the gastricsins (EC3.4.23.3). Progastricsin or pepsinogen C (PGC) is converted to gastricsin by removal of the 43 amino-terminal residues of the prosegment. The resulting mature gastricsin has 329 amino acid residues. The sequence of human PGC has been determined independently in two laboratories by nucleotide sequencing of the gene (2) and of cDNA clones (3).

Crystallographic Studies of an Activation Intermediate of Human Gastricsin

The human digestive enzyme progastricsin (hPGC) is an aspartic proteinase zymogen that is synthesized as an inactive precursor, having a positively charged and inhibitory N-terminal prosegment of 43 residues (Ala1p to Leu43p; the “p” suffix refers to the prosegment). Conversion of progastricsin to mature gastricsin occurs upon the lowering of pH, and involves conformational changes that uncover the active site.1 The initial hydrolytic event is the uni-molecular, auto-catalytic cleavage of the peptide bond between Phe26p and Leu27p in the prosegment.1,3 Further proteolytic processing is then initiated, ultimately resulting in the removal of the entire prosegment and formation of the mature enzyme. Our lab has previously determined the crystal structure of hPGC.2 The structure of mature gastricsin is currently undetermined, although it is expected to resemble human pepsin and the other aspartic proteinases.