Pepsinogens and Pepsins from House Musk Shrew, Suncus murinus: Purification, Characterization, Determination of the Amino-Acid Sequences of the Activation Segments, and Analysis of Proteolytic Specificities

Abstract

Three pepsinogens, namely, pepsinogens A, C-1, and C-2, were purified from gastric mucosa of adult house musk shrew (Suncus murinus) by conventional chromatographic and gel filtration procedures. The molecular masses were 40, 39, and 41 kDa for pepsinogens A, C-1, and C-2, respectively. Pepsinogen C-2 contains an Asn-linked carbohydrate chain(s) of about 2 kDa. Each pepsinogen was converted to pepsin through an intermediate form under acidic conditions. By NH2-terminal sequence analysis of these protein species, the amino acid sequences of activation segments (proparts) of pepsinogens A and C-1 were determined to be LYKVPLVKKKSLRQNLIENGLLKDFLAKHNVNPASKYFPTE and KVTKVTLKKFKSIRENLREQGLLEDFLKTNHYDPAQKYHFGDF, respectively. The similarity of these two sequences is nearly 50%. Each pepsin cleaved preferentially peptide bonds between hydrophobic and aromatic amino acids, or bonds on either side of these amino acids. Although each activation segment had several sites susceptible to pepsin action, activation proceeded by limited cleavages of the segment, presumably due to the steric inflexibility of the segment in native pepsinogen. The activity of pepsin A was inhibited completely in the presence of a more than equimolar amount of pepstatin, while a hundred-molar excess amount of pepstatin was needed for the complete inhibition of the activity of pepsins C-1 and C-2.

Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development

“…Pepsinogen A and progastricsin are mainly expressed in adult vertebrates, although their relative levels differ greatly between animals ( fig.1). Both types of pepsinogen are found in primates [12], insectivores [29], and even-toed ungulates [27,30], although type A pepsinogens predominate in most of these mammals. In the rabbit [13] and carnivores such as the dog [31], pepsinogens A are nearly exclusive components, and progastricsins have not been isolated from these mammals.…”

“…Apart from two different activation pathways, two different cleavage reactions are possible for activation: in- Figure 4. The primary cleavage sites upon activation of various pepsinogens are summarized based on the results of pepsinogens A from human [80,87], monkey [12,73,83], pig [74,76], cattle [142], bear [69], house musk shrew [29], rabbit [13], and chicken [81]; cattle pepsinogen B [38]; progastricsins from human [77], monkey [12,83], pig [143], house musk shrew [29], and frog [22]; cattle prochymosin [78]; and tuna pepsinogens [144]. The residues in the activation segment are numbered according to the porcine pepsinogen A sequence and characterized by the suffix p. Residues in filled boxes are conserved in these sequences.…”

“…Like pepsin A, gastricsin has high proteolytic activity at acidic pH. In several mammalian species, it is known to be maximally active around pH 3 against hemoglobin, a slightly higher pH value than that observed for pepsin A, and its specific activity is about twofold higher [29,30,92]. This proteolytic specificity has not been studied as extensively as that of pepsin A, but comparison of human pepsin A and gastricsin showed that although the latter has generally similar substrate specificity, it prefers Tyr at the P 1 position and thus cleaves Tyr-X bonds preferentially ( fig.…”

Phylogenetic Position of Eulipotyphla Inferred from the cDNA Sequences of Pepsinogens A and C

“…Multiplicity of pepsinogen A varies among mammals. Mammals showing high multiplicity include human (Athauda et al, 1989), monkey (Kageyama, 1994), and rabbit (Kageyama et al, 1990), whereas those showing low multiplicity include the house musk shrew (Narita et al, 1997). Mammals used in the present study are included in the latter group.…”

Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts,Cynops pyrrhogaster

“…Mammalian pepsin C1s are known to be less sensitive to the inhibitor than pepsin A. For example, mammalian pepsin C1s, such as those from the house musk shrew (29), goat (30) and new world monkey (32), require a 100-molar excess of pepstatin for complete inhibition. Thus, the inhibitory properties of newt PgBC are similar to those of pepsin C1s in mammals.…”