Acidic Proregion Research Articles

Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli.

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.

A novel Kex2 enzyme can process the proregion of the yeast alpha-factor leader in the endoplasmic reticulum instead of in the Golgi

The prepro sequence of the yeast prepro-α-factor, usually referred to as the α-factor leader, has often been used for the efficient secretion of heterologous proteins from the yeast Saccharomyces cerevisiae . The α-factor leader consists of a 19-amino acid N-terminal pre or signal sequence followed by a 66-amino acid proregion. After removal of the signal sequence during membrane translocation, the proregion is cleaved from the precursor protein by the Kex2 endoprotease only in a late Golgi compartment. Here we report that a modified Kex2 enzyme, containing at the C-terminus the HDEL tetrapeptide, cleaves the proregion from the α-factor leader-human insulin like growth factor-1 fusion protein in the endoplasmic reticulum. The processing of pro-proteins earlier in the secretion pathway could be helpful in defining the cellular function of the proregions present naturally in various eucaryotic precursor proteins.

Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence.

Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.

Human renin biosynthesis and secretion in normal and ischemic kidneys.

The pathway of renin biosynthesis and secretion in normal and ischemic human kidneys has been investigated by pulse-labeling experiments. The results indicate that in normal human kidney, preprorenin is rapidly processed to 47-kDa prorenin. Microradiosequencing showed that this molecule was generated by cleavage between Gly-23 and Leu-24, yielding a 43-amino acid proregion. Analysis of prorenin secreted by the kidney tissue yielded an identical sequence, indicating that prorenin is secreted without any further proteolysis. An examination of the kinetics of processing and secretion suggested that a majority of the newly synthesized prorenin is quickly secreted, while only a small fraction is processed intracellularly to the mature renin. The differences in secretion kinetics between prorenin and mature renin and the selective inhibition of prorenin secretion by monensin suggest that they are secreted independently via two pathways: a constitutive pathway probably from the Golgi or protogranules that rapidly release prorenin and a regulated pathway that secretes mature renin from the mature granules. A comparison of the kinetics of processing between normal and ischemic tissues suggests that renal ischemia leads to an overall increase in the rate of processing of prorenin to mature renin. In addition, prolonged biosynthetic labeling of renin in the ischemic kidney yielded two smaller molecular weight immunoreactive forms suggestive of renin fragments that may be degradative products. These fragments were not detected in normal kidney tissue labeled for similar lengths of time.

Cloning and characterization of a mouse cysteine proteinase.

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.

Structure and Multistep Activation of the Precursors of Peptides from Honeybee Venom Glands and Frog Skin

Publisher Summary This chapter discusses structure and multistep activation of the precursors of peptides from honeybee venom glands and frog skin. It presents studies on the biosynthesis of peptides in honeybee venom glands and amphibian skin. The main constituent of honeybee venom is melittin, is a lytic peptide comprising 26 amino acids. In vitro translation of melittin mRNA isolated from venom glands of young queen bees yields prepromelittin that contains 70 residues. Starting from the amino end, four different parts can be discerned in the structure of this polypeptide: (1) a preor signal peptide of 21 mostly hydrophobic residues, (2) an acidic proregion of 22 amino acids with either proline or alanine at even-numbered positions, (3) the 26 amino acids of melittin, and (4) an extra glycine residue at the carboxyl end. The liberation of melittin from its precursor is a multistep process, which involves three types of enzymatic reactions. These are the endoproteolytic cleavage of the signal peptide, the formation of the carboxy-terminal amide with concomitant loss of the terminal glycine, and the stepwise activation of promelittin by an exoprotease. The chapter describes the three enzymatic reactions in detail. From amphibian skin, a variety of peptides have been isolated that were found to be similar or identical to mammalian hormones or neuropeptides. The chapter describes he biosynthesis of frog skin peptides as a promising model system for the formation of homologous peptides in the mammalian gastrointestinal tract and nervous system.