Calcium‐dependent KEX2‐like protease found in hepatic secretory vesicles converts proalbumin to albumin

Brennan, Stephen O.; Peach, Robert

doi:10.1016/0014-5793(88)80819-6

Cited by 73 publications

(29 citation statements)

References 21 publications

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“…Since the discovery of the first mammalian KEX2-like protease in hepatic secretory vesicles [1] a series ofcDNA homologs (furin PC2, PCI/3, PC4, PC6 and PACE4) of this yeast convertase has now been identified in different mammalian tissues [2][3][4][5][6][7][8][9][10]. Members of this emerging family of Ca z+ -dependent serine proteases vary in their site of expression and in the basic sequence that they recognise.…”

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confidence: 99%

Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P₂ and P′₁ sites

Brennan

Nakayam

1994

FEBS Letters

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AlmtractProalbumin is the principal substrate of the in situ hepatic convertase. Here we investigated the specificity of furin using synthetic peptides based on the N-terminal sequeaee of human proalbumin. The propeptide was rapidly cleaved from the normal ((-6)RGVFRR(-1)DAHKSEAVW(+ 9)) peptide but as expected, there was no cleavage of the proalbumin Lille analogue with a -2 His (-2H). Surprisingly, the effect of this substitution could not be corrected by introducing a -4 Arg (-4R-2H). In contrast, the peptide -4R-2A was an excellent substrate being cleaved five times faster than normal, indicaabting that His is not allowed as an P2 residue. Replacement of the -4 Val by Glu supported the expected importance of a positive charge at P4 as the cleavage rate dropped to 10% of normal after this substitution. The -6 Arg makes a small contribution to cleavage, its replacement by Aia decreased the cleavage rate to 60% of normal. The Lys-Arg propeptide was almost as good a substrate as the normal Arg-Arg peptide, but the introduction of a Lys at P'1 totally abolished processing. The exclusion of P't positive charges would be an important requirement for preventing aberrant cleavage in the middle of tetrabasic sequences.

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Section: Introductionmentioning

confidence: 99%

Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P₂ and P′₁ sites

Brennan

Nakayam

1994

FEBS Letters

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“…The hypothetical roles of this "Kex2 subfamily" in processing secretory precursors in animal cells are under investigation in many laboratories. Properties of the partially purified yeast enzyme have served as a point of comparison with activities that cleave proinsulin (11) and proalbumin (12). Analysis of purified Kex2 protease should help define the nature and structural basis of substrate specificity in the Kex2 subfamily, the biological roles of Kex2 and its homologues, and the pathway of biosynthesis and posttranslational modification of these enzymies.…”

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Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease.

Brenner

Fuller

1992

Proc. Natl. Acad. Sci. U.S.A.

177

145

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The prohormone-processing Kex2 protease of the budding yeast Saccharomyces cerevisiae can be converted from an intracellular membrane protein to a soluble, secreted, and active form by deletion of the transmembrane domain and C-terminal tail. One such molecule was purified to near homogeneity from the culture medium of an overexpressing yeast strain. Amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially autoproteolytic cleavage at Lys'"-Arg, prior to the domain homologous to subtilisin, followed by trimming of Leu-Pro and Val-Pro dipeptides by the Stel3 dipeptidyl aminopeptidase. Kinetic parameters were examined using fluorogenic peptidylmethylcoumarin amide substrates. Initial burst titration indicated that the preparation was entirely active. Measurements of dependence of activity on pH yielded a simple curve suggesting titration of a single ionizable group. Activity was half-maimal at pH 5.7 and nearly constant from pH 6.5 to 9.5. Discrimination between substrates was as great as 360-fold in Km and 130-fold in kt. Substrates with a Lys-Arg dipeptide preceding the cleaved bond were preferred, having ktlKm values up to 1.1 x 107 sec-1 M-'. The enzyme cleaved substrates having Arg-Arg, Pro-Arg, Ala-Arg, and Thr-Arg with increased Km but with unchanged k,,t. In contrast, the enzyme displayed a dramatically lower kt for a Lys-Lys substrate with a smaller increase in K. Thus the two residues preceding the cleaved bond may play distinct roles in the selectivity ofbinding and cleavage of probormone substrates.The Saccharomyces cerevisiae Kex2 protein, a Ca2+-dependent serine protease, is the only enzyme proven by genetic and biochemical evidence to cleave a prohormone (pro-a-factor) at pairs of basic residues in the eukaryotic secretory pathway (1-3). The high specificity of Kex2 protease, homologous to the subtilisin family (4, 5), contrasts with the broad specificity of previously known members of the family, all of which are degradative enzymes. Kex2 and its mammalian homologues furin (5, 6), PC2 (7,8) MATERIALS AND METHODS Expression System. ss-Kex2 was purified from the culture media of strains CB023 and KRY77-3B carrying plasmid pG5-KEX2AC3 (Fig. 1)

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“…These cleavages occur on either side of dibasic pairs of amino acids, such as Arg-Arg or Lys-Arg, or on the COOH- (Benoit et al, 1988;Fung et al, 1984) or NH,-terminal side (Devi et al, 1989;Civelli et al, 1985) of monobasic residues, usually Arg, in a number of processed proteins. Enzymes catalyzing these cleavages have been described in a late Golgi compartment of yeast (the Kex2 protease, Redding et al, 1991;Wilcox and Fuller, 1991), in rat liver Golgi membranes (Mizuno et al, 1989;Brennan and Peach, 1988) and in the Golgi apparatus of rat neural (Lepage-Lezin et al, 1991) and pituitary cells (Schnabel et al, 1989). As p l4GalT contains several Arg residues in ifs putative stein region, it was certainly conceivable that the proteolytic cleavages observed in the purified proteins might represent normal processing events, and that the protein might be bound to Golgi membranes by some other means.…”

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Proteins of the Golgi apparatus

Bendiak

Ward

Simpson

1993

European Journal of Biochemistry

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The Golgi marker enzyme, UDP‐galactose: N‐acetylglucosamine β1‐4galactosyltransferase (β1‐4GalT) was purified 44300‐fold in its intact, membrane‐bound form from rat liver membranes. The protein was isolated from detergent extracts as a high‐Mr form, having a Stokes radius approximating a globular protein of Mr 440000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an Mr near 51000, and does not have intermolecular disulfide crosslinks. N‐terminal sequencing of the enzyme demonstrated that it contains an N‐terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5′ end of the mRNA [Russo, R. N, Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324–3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N‐terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low‐Mr forms which were fully catalytically active and which, upon sequencing, were missing a 66‐amino‐acid stretch from the N‐terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with Mr near 50000. The N‐terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non‐protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

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Calcium‐dependent KEX2‐like protease found in hepatic secretory vesicles converts proalbumin to albumin

Cited by 73 publications

References 21 publications

Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P₂ and P′₁ sites

Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P₂ and P′₁ sites

Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease.

Proteins of the Golgi apparatus

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Calcium‐dependent KEX2‐like protease found in hepatic secretory vesicles converts proalbumin to albumin

Cited by 73 publications

References 21 publications

Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P2 and P′1 sites

Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P2 and P′1 sites

Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease.

Proteins of the Golgi apparatus

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Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P₂ and P′₁ sites

Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P₂ and P′₁ sites