PubMed Health⌕ Search

Biomedical subjects

G Koelsch

Publications and source records attributed to G Koelsch.

27 records · Page 2Linked to original sources
PubMed1994

Multiple functions of pro-parts of aspartic proteinase zymogens.

The importance of aspartic proteinases in human pathophysiology continues to initiate extensive research. With burgeoning information on their biological functions and structures, the traditional view of the role of activation peptides of aspartic proteinases solely as inhibitors of the active site is changing. These peptide segments, or pro-parts, are deemed important for correct folding, targeting, and control of the activation of aspartic proteinase zymogens. Consequently, the primary structures of pro-parts reflect these functions. We discuss guidelines for formation of hypotheses derived from comparing the physiological function of aspartic proteinases and sequences of their pro-parts.

Amino Acid Sequence↗
PubMed1994

Proteolytic processing mechanisms of a miniprecursor of the aspartic protease of human immunodeficiency virus type 1.

The infectivity of the human immunodeficiency virus (HIV) depends upon correct proteolytic processing of viral polyprotein precursors, the Pr55gag and Pr160gag-pol polyproteins. The processing is mediated spontaneously by the viral protease unit (PR) contained within the Pr160gag-pol precursor. However, little is known about the mechanism of this process. The expression in Escherichia coli and the isolation of a 14-kDa HIV-1 PR "miniprecursor" with Ala28 mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of the protease. The miniprecursor is active against a synthetic peptide substrate, and its specific activity is near that of the mutant mature protease. The rate of conversion of radiolabeled precursor to mature protease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion rate constant, kapp, is dependent on miniprecursor concentration indicating a second-order reaction and suggesting an interdimeric processing mechanism. A significant first-order rate constant is observed when the plot of kapp versus initial precursor concentration is extrapolated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The presence of both mechanisms is an advantage for the virus to ensure processing under various conditions.

Amino Acid Sequence↗
PubMed1993

A hypothesis for the HLA-B27 immune dysregulation in spondyloarthropathy: contributions from enteric organisms, B27 structure, peptides bound by B27, and convergent evolution.

Several human rheumatic diseases occur predominantly in persons who carry the histocompatibility (HLA) class I allele B27. They have also been related to Gram-negative enteric microorganisms. In addition, the recent recovery of peptides bound to B27 has allowed an understanding of the structural requirements for their binding. Using the accumulated data base of protein sequences, we have tested a series of hypotheses. First, we have asked whether the primary amino acid sequence of the hypervariable regions of HLA-B27 shares short sequences with the proteins of Gram-negative enteric bacteria. The data demonstrate that, unique among the HLA-B molecules, the hypervariable regions of HLA-B27 unexpectedly share short peptide sequences with proteins from these bacteria. Second, we have asked whether the enteric proteins tend to satisfy the structural requirements for peptide binding to B27 in those regions of the sequence shared with B27. This hypothesis also tends to be true, especially in an allelically variable part of the B27 sequence which is predicted to bind B27 if it were to be presented as a free peptide. We conclude that HLA-B27 and enteric Gram-negative bacteria have undergone a previously unappreciated form of convergent evolution which may be important in the process leading to these rheumatic diseases. Moreover, the regions of the enteric bacterial proteins which are contiguous with the short sequences shared with B27 tend to have structures which are also predicted to bind B27. These observations suggest a mechanism for autoimmunity and lead to the prediction that the B27-associated diseases are mediated by a subset of T-cell receptors, B27, and the peptides bound by B27.

Amino Acid Sequence↗
PubMed1993

Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties.

A cDNA fragment which encodes the zymogen of canditropsin, the extracellular aspartic protease from the yeast Candida tropicalis (Togni,G., Sanglard, D., Falchetto, R., and Monod, M. (1991) FEBS Lett. 286, 181-185) was cloned into a T7 expression vector for the synthesis of the recombinant zymogen in Escherichia coli. Recombinant canditropsinogen (Ctg), which was expressed as inclusion bodies in the cytosol of E. coli, was refolded by dialysis from an 8 M urea solution and purified to homogeneity using chromatographies on Sephacryl S-300 and on MonoQ columns. The purified Ctg was converted into canditropsin by either acid activation or trypsin conversion. The specificity of the resulting recombinant canditropsin toward polypeptide substrates is significantly different from other aspartic proteases. Canditropsin hydrolyzes oxidized insulin B chain between Ala-Leu and many other minor cleavage sites. Canditropsin also hydrolyzes keratin and collagen, which are components of connective tissues known to be hydrolyzed by canditropsin during Candida infections. Canditropsin was strongly inhibited by the universal aspartic protease inhibitor pepstatin (Ki = 1.75 x 10(-8) M) and inactivated by two aspartic protease inactivators, DAN and EPNP. Canditropsin is weakly inhibited by leupeptin and antipain, with an apparent Ki of 1.74 x 10(-4)M and 1.5 x 10(-5) M, respectively.

Amino Acid Sequence↗
PubMed1992

Enzymic activities of two-chain pepsinogen, two-chain pepsin, and the amino-terminal lobe of pepsinogen.

In order to study the relationships of aspartic proteases, we have modified pepsin, a single-chain eukaryotic enzyme, to a two-chain heterodimer, which resembles aspartic proteases from retrovirus, including human immunodeficiency virus. Two fragments of pepsinogen, residues 1P-172 and 173-326, were expressed separately in Escherichia coli. Mixtures of chains were refolded from urea solutions to generate an active two-chain pepsinogen, which was converted to two-chain pepsin in acid solutions. The intramolecular and bimolecular activation constants (k1 and k2) of two-chain pepsinogen are about 1.5-fold and one-sixth, respectively, of those for pepsinogen. Structural evidence suggests that the faster k1 of two-chain pepsinogen is due to decreased interaction of the propeptide with the pepsin moiety, implying that the rate-limiting step in the intramolecular activation of pepsinogen is the "conformational dissociation" of its propeptide. Two-chain pepsin has the same Km but only one-sixth of the kcat of pepsin. Both pepsinogen chains are capable of independent refolding. The refolding of the NH2-terminal chain, which contains the propeptide and the NH2-terminal lobe, generated a small amount of proteolytic activity which is likely derived from the homodimer of the NH2-terminal lobe. It has been postulated that mammalian aspartic proteases, which contain two structurally homologous lobes, are derived in evolution from a homodimer enzyme by gene duplication and fusion (Tang, J., James, M. N. G., Hsu, I.-N., Jenkins, J. A., and Blundell, T. L. (1978) Nature 271, 618-621). The observation of the homodimer activity of the NH2-terminal lobe of pepsinogen suggests that the interface of the lobes is conservative in evolution.

Base Sequence↗
PubMed1992

The high-resolution crystal structure of porcine pepsinogen.

The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 A. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of beta-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (1P-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family.

Amino Acid Sequence↗
PubMed1991

Mapping and molecular modeling of a recognition domain for lysosomal enzyme targeting.

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose-6-P residues. Previously, we generated a lysosomal enzyme recognition domain by substituting two regions (lysine 203 and amino acids 265-292) of the lysosomal hydrolase cathepsin D into a related secretory protein glycopepsinogen. When expressed in Xenopus oocytes, the oligosaccharides of the chimeric protein were efficiently phosphorylated (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). In the current study, incremental substitutions of cathepsin D residues into glycopepsinogen and alanine-scanning mutagenesis were utilized to define the recognition domain more precisely. A computer-generated model of the cathepsin D/pepsinogen chimeric molecule served as a guide for mutagenesis and for the interpretation of results. These studies indicate that the recognition domain is a surface patch that contains multiple interacting sites. There is a strict positional requirement for the lysine residue at position 203.

Amino Acid Sequence↗
PubMed1991

Recombinant rhizopuspepsinogen. Expression, purification, and activation properties of recombinant rhizopuspepsinogens.

A cDNA clone, which contained the complete rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of rhizopuspepsinogen (Rpg). Recombinant Rpgs which were expressed in the cytosol of E. coli as inclusion bodies (cRpg and tRpg) were not active. After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity. The third zymogen, pRpg, which was secreted to the periplasmic space of E. coli with an omp leader, was fully active and also was purified. The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture). Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively. tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture. This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction. All of these Rpgs spontaneously converted to rhizopuspepsin in solutions of pH less than 5. Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis. At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens. For pRpg and tRpg, kinetic data demonstrated that the Rpg to rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2. The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively. The activation rate decreased as pH was raised above pH 2. At pH greater than 3.0, rhizopuspepsin-catalyzed, second-order activation also takes place. Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen. These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen. Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become rhizopuspepsin before secretion.

Amino Acid Sequence↗