From the Department of Biochemistry, University of
Kentucky, Lexington, Kentucky 40536-0084, ¶ Mass Spectrometry
Facility, University of Kentucky, Lexington, Kentucky
40506-0286, and the § Department of Biophysics,
Escola Paulista de Medicina, Sao Paulo 04034, Brazil
Received for publication, September 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant rat insulysin was shown to cleave the
internally quenched fluorogenic peptide
2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K
bond, exhibiting a Km of 13 µM and a
Vmax of 2.6 µmol min Insulysin (insulin-degrading enzyme; EC 3.4.24.56) was first
described as a proteolytic enzyme capable of degrading insulin (1). The enzyme is primarily located in the cytosol and peroxisomes (2), although its presence on the cell membrane (3, 4) and its
secretion (5) have recently been reported. Although insulysin has the
highest affinity for insulin, the enzyme has been shown to cleave a
number of other physiological peptides in vitro including
glucagon (6), insulin-like growth factors I and II (7), atrial
natriuretic peptide (8), and transforming growth factor- Insulysin was shown to be identical to an enzymatic activity
referred to as Insulysin is a 110-kDa zinc metallopeptidase member of the invercinzin
family of proteases, characterized by an inverted active site motif,
HXXEH, relative to the more common HEXXH motif.
The two histidines in this motif are ligands for the active site zinc atom, and the glutamate serves either as a nucleophile attacking the
susceptible peptide bond or more likely as a general acid catalyst
facilitating the attack of a water molecule on the scissile bond (18).
The functionality of these three residues has been established by
site-directed mutagenesis (18-20).
The specificity of insulysin toward peptide bond cleavage is complex
and not fully understood. Authier et al. (6) examined the
cleavage sites in a variety of peptides and concluded that the enzyme
exhibited a preference for basic (arginine) or large hydrophobic
(phenylalanine, leucine, tyrosine) amino acids to occupy the
carboxyl (P1) side of the cleavage site. We
previously conducted a similar analysis of the substrate specificity of
the enzyme and also concluded that the enzyme exhibits a preference, but not absolute specificity, for cleavage at hydrophobic and basic
residues (13). However, we suggested that the specificity of insulysin
is directed at the amino side (P1') rather than the carboxyl side (P1) of the bond cleaved. It has also been
proposed that the specificity of the enzyme is governed by the
three-dimensional structure of the substrate (2). This is not
inconsistent with the specificity being directed at basic and
hydrophobic residues, because it has been observed that substrates bind
in an extended In this study we have introduced the use of internally quenched
fluorogenic peptides as substrates for insulysin. We have used these
peptides to further probe the specificity of the enzyme. The results
indicate the importance of subsite interactions in determining cleavage
sites and further support the suggestion that insulysin specificity is
directed at the amino side of hydrophobic and basic residues.
Expression and Purification of Recombinant Insulysin--
A rat
insulysin cDNA (3), kindly provided by Dr. Richard Roth of Stanford
University, was subcloned into the pFASTBAC vector (Life Technologies,
Inc.) using BamHI and XhoI restriction sites such
that a His6 affinity tag became fused to the N terminus of the protein. The insulysin portion of the fusion protein started at
methionine 42, which corresponds to the second putative start site in
the coding region. This latter site is believed to be the one utilized
in vivo (21). Generation of recombinant baculovirus and
expression of recombinant insulysin in Sf9 cells were done according to the manufacturer's instructions. For the purification of
recombinant insulysin, a 1:10 (w/v) suspension of Sf9
cells expressing the enzyme was prepared in 100 mM
potassium phosphate buffer, pH 7.3, containing 1 mM
dithiothreitol (K-PO4/dithioerythritol buffer). The
cells were sonicated using a Branson sonifier (setting 3 at 30%) 10 times, each time for 1 s. The sonicate was centrifuged at
75,000 × g for 20 min to pellet the membrane fraction,
and the supernatant containing insulysin was loaded onto a 5-ml
nickel-nitrilotriacetic acid column (Qiagen) that had been
equilibrated with the K-PO4/dithioerythritol buffer. After
extensive washing of the column with starting buffer and then with 20 mM imidazole buffer, pH 7.3, the enzyme was eluted with 500 mM imidazole buffer, pH 7.3. From a 100-ml culture ~2 mg
of purified enzyme was obtained. The purified enzyme was essentially homogeneous as judged by SDS-polyacrylamide gel electrophoresis (Fig.
1).
HPLC1
Assays--
During purification, the activity of insulysin was assayed
by measuring the disappearance of Fluorogenic Assays and Determination of Kinetic
Parameters--
The reaction of fluorogenic substrates containing an
N-terminal 2-aminobenzyl (Abz) group and a C-terminal
ethylenediamine-2,4-dinitrophenyl group (EDDnp) was measured by the
increase in fluorescence that occurs upon cleavage of any peptide bond
and the separation of the quenching EDDnp group from the fluorescent
Abz group. Peptides were synthesized and purified by HPLC as described
previously (22) and prepared as 1 mM stock solutions in
20% dimethylformamide. The peptide concentration was calculated from
the absorbance at 357 nm ( Determination of Sites of Peptide Cleavage--
Reaction
mixtures containing 40 µM fluorogenic peptide were
allowed to react for varying time periods and stopped as noted above.
The reaction mixture was then subjected to HPLC using a gradient of 5%
acetonitrile in 0.1% aqueous trifluoroacetic acid to 50% acetonitrile
in 0.1% aqueous trifluoroacetic acid. Product peaks were either
collected and identified by matrix-assisted laser desorption ionization
time of flight mass spectrometry or by comparison of the retention
times of products to those of previously identified peptides. In some
cases product identification was verified by mixing a known product
with the unknown and demonstrating co-chromatography in the HPLC
gradient system.
Insulysin activity has generally been assayed with iodinated
insulin as substrate. This assay involves reaction of the enzyme with
this substrate, followed by precipitation of unreacted insulin with
trichloroacetic acid and subsequent measurement of the radiolabeled products in the supernatant. Although this assay is rather sensitive, it has several disadvantages. It is cumbersome, the products
formed are not determined during the reaction, it requires several
cleavages of the insulin molecule to release soluble products, it is a
discontinuous assay, and it requires handling radioactive material. An
alternative assay was developed (13) in which the disappearance of
another insulysin substrate, In an attempt to develop a simpler continuous assay for the enzyme, we
tested as a substrate the internally quenched fluorogenic peptide
Abz-GGFLRKVGQ-EDDnp, which contains a central core of both hydrophobic
and basic residues, which are cleavage sites in the substrates
1
mg
1. Derivatives of this peptide in which the
P2 leucine or the P2' valine were replaced
with other residues were used to probe the subsite specificity of the
enzyme. Varying the P2 residue produced a 4-fold range in
Km and a 7-fold range in
kcat. The nature of the P2 residue
had a significant effect on the site of cleavage. Leucine, isoleucine,
valine, and aspartate produced cleavage at the R-K bond. Asparagine
produced 36% cleavage at the N-R bond and 64% cleavage at the R-K
bond, whereas with alanine or serine the A-R and S-R bonds were the
major cleavage sites. With tyrosine, phenylalanine, methionine, or
histidine representing the varied residue X, cleavages at
F-X, X-R, and R-K were seen, whereas with
tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable
P2' residues produce less of a change in both Km and kcat and have little
influence on the cleavage site. Exceptions are phenylalanine,
tyrosine, leucine, and isoleucine, which in addition to producing
cleavage at the R-K bond, produce significant cleavage at the L-R
bond. Alanine and tyrosine were unique in producing cleavage at the
F-L bond. Taken together, these data suggest that insulysin
specificity is directed toward the amino side of hydrophobic and basic
residues and that the enzyme has an extended substrate binding site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(9). The
finding of a variety of substrates for the enzyme, as well as its
presence at high levels in insulin-insensitive cells, suggest that
insulysin has a variety of physiological functions. Those proposed
include processing of insulin for antigen recognition (10), regulation
of the multicatalytic proteinase (11), and modulation of steroid
receptor action (12).
-endorphin-generating enzyme, an enzyme that converts
-endorphin to
-endorphin (
endorphin 1-17) and
endorphin 1-18 (13). In that study GRF, dynorphin B 1-13,
dynorphin A 1-17, and pancreastatin 1-49 were shown also to be
substrates. Recent interest in insulysin stems from its ability to
degrade the amyloid peptides A
1
40 and A
1-42 (5, 14-16) and
its possible role as an enzyme involved in the clearance of amyloid peptides in the brain. Decreases in insulysin activity in the brains of
Alzheimer's patients have been suggested to contribute to the
progression of this disease (17).
-like structure to many proteases.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
SDS-polyacrylamide gel electrophoresis
analysis of purified rat insulysin. Insulysin (15 µg) purified
by affinity chromatography as described under "Materials and
Methods" was analyzed by SDS-polyacrylamide gel electrophoresis on a
7.5% gel stained with Coomassie Blue.
-endorphin using reverse phase HPLC as described previously (13), except that isocratic elution was
employed. A 100-µl reaction mixture containing 40 mM
potassium phosphate buffer, pH 7.3, 30 µM
-endorphin,
and enzyme was incubated for 15 min at 37 °C. The reaction was
stopped by the addition of 10 µl of 5.5% trifluoroacetic acid,
producing a final concentration of 0.5%. The reaction mixture was
loaded onto a C4 reverse phase HPLC column (Vydac,
Hisperia, CA) and separated isocratically at 32% acetonitrile in 0.1%
aqueous trifluoroacetic acid. The
endorphin, which eluted at ~8
min, was detected by absorbance at 214 nm using a Waters detector and
quantitated from its peak area.
= 10 mM). Reaction
mixtures contained the fluorogenic peptide substrate, insulysin, and 20 mM potassium phosphate buffer, pH 7.3. The reaction was
initiated by the addition of enzyme and monitored continuously on a
Hitachi model F2000 spectrofluorometer equipped with a chart recorder.
Excitation and emission wavelengths were 318 and 418 nm, respectively.
The relationship between fluorescence change and peptide concentration
was determined by measuring the total fluorescence change that occurred
upon complete hydrolysis of the peptide with trypsin (22). Kinetic data
were fit to computer programs of Cleland (23).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-endorphin, is measured by HPLC. This
assay has the advantage that it does not require a radioactive
substrate, but it has the disadvantages that it is also a discontinuous
assay, it follows substrate disappearance rather than product
appearance, and it requires ~1 h for each HPLC run. In this study the
use of an isocratic system rather than a gradient system to follow
-endorphin disappearance reduced the assay time from over an hour to
less than 10 min.
-endorphin and insulin. Cleavage of any peptide bond in this
substrate produces an increase in fluorescence as the quenching of the
N-terminal fluorescent Abz group by the C-terminal EDDnp group is
relieved (24, 25). We found this peptide to be cleaved by purified
recombinant insulysin, yielding a Km of 21 µM and a Vmax of ~0.2
µmol min
1 mg
1 of enzyme. This can
be compared with
-endorphin, which exhibits a Km
of 13 µM and a Vmax of 2.6 µmol min
1 mg
1. Cleavage of
Abz-GGFLRKVGQ-EDDnp occurred at a single site, the R-K bond (Fig.
2).
View larger version (24K):
[in a new window]
Fig. 2.
HPLC chromatograms showing the hydrolysis
products generated from internally quenched peptides. Reaction
mixtures containing 40 µM internally quenched peptides
were reacted with rat insulysin in 50 mM potassium
phosphate buffer, pH 7.3. Reaction products were separated by gradient
HPLC on a Vydac C4 reverse phase column as described under
"Materials and Methods." Product peaks were collected and
identified by either comparison to standards or by mass spectrometry.
Shown are the products derived from Abz-GGFXRKVGQ-EDDnp
where X = leucine (A), X = tryptophan (B), and X = phenylalanine
(C). The major peak eluting between 35 and 40 min represents
unreacted substrate.
The ability to use quenched fluorogenic peptides as substrates for insulysin permitted an assessment of the effect of varying the amino acid residue at specific positions on the hydrolysis of peptides by this enzyme. Shown in Table I is the effect of varying amino acids in the P2 position.2 It can be seen that variable P2 residues affect the apparent kcat, with a 7-fold variation in this parameter observed, whereas Km varies less than 4-fold; and changes in kcat/Km more or less parallel kcat. As shown in Table I and exemplified in Fig. 2, there is a rather profound effect of the P2 residue on the site of bond cleavage. With the aliphatic residues leucine, isoleucine, valine, and aspartate, cleavage occurs exclusively at the R-K bond. With asparagine, cleavage at the N-R bond is observed about one third of the time, whereas with the small side chains of alanine and serine, the major cleavage sites are the A-R and S-R bonds, respectively. With tyrosine, phenylalanine, methionine, or histidine corresponding to the variable amino acid, three cleavage sites are observed, which are at the F-X bond, the X-R bond, and the R-K bond. The extent of cleavage at each of these sites is dependent on the particular amino acid residue present. With tryptophan, cleavage at the R-K bond does not occur; however, cleavage is seen at the F-W and W-R bonds at about equal frequency. It should be noted that product analysis revealed only the intact N- and C-terminal fragments resulting from the indicated cleavage sites. No fragments corresponding to secondary cleavages were observed.
|
These data indicate that there are multiple alignments permitted for substrate binding. Subsite interactions between the enzyme and the substrate govern these alignments, which in turn determine which bond is cleaved. Within the sequences tested, the bulky hydrophobic amino acids leucine, isoleucine, and valine, as well as aspartate, appear to be accommodated only as a P2 residue leading to cleavage exclusively at the R-K bond. Leucine produces the highest kcat value when acting as a P2 residue (Table II). At the other extreme, tryptophan does not appear to occupy the P2 position. Two nonexclusive explanations that can account for this observation are possible. Tryptophan may fit poorly into the S2 subsite of the enzyme. Additionally, tryptophan may be favored as both a P1 and P1' residue and thus within the sequence FWRKV cleavages at F-W or W-R occur much faster than at the R-K bond. All other residues examined appear to be capable of occupying the P2 position but have a relatively small influence on the rate of cleavage at the R-K bond (Table II). This suggests that there is no correlation between cleavage at a particular bond and kcat.
|
As shown in Tables I and II, within the sequence FXRKV, where X is the variable amino acid, only a limited number of residues were found to serve as P1' residues. These were the aromatic amino acids and methionine. Of these, tyrosine produces the highest kcat for cleavage at the F-X bond. Within this varied sequence, the enzyme accepts a variety of amino acids as P1 residues, with the rate of cleavage exhibiting an ~10-fold variation dependent on the specific residue occupying this position. Leucine, valine, isoleucine, and aspartate, although not found as P1 residues, were found as P2 residues. The variety of amino acid types that can occupy the P1 position and the observation that insulysin appears to favor cleavage at hydrophobic and basic residues (6, 13) make it less likely that insulysin specificity is directed at the P1 residue.
The effect of varying the P2' residue is shown in Table III. With the exception of aspartate, which is the least favored residue in the P2' position, there is less than a 5-fold effect of variable P2' residues on the apparent kcat, whereas Km varies only ~3-fold. When one considers the rates for cleavage specifically at the R-K bond, excluding aspartate, an ~8-fold range of kcat values becomes evident. Histidine and serine are the preferred residues at this site, with no obvious pattern of discrimination among other types of side chains. There is also an effect of the P2' residue on the site of cleavage. The aromatic amino acids phenylalanine and tyrosine and the hydrophobic amino acids leucine and isoleucine produce significant cleavage at the L-R bond and thus act as P3' residues. Alanine, but not serine, and to a lesser extent tyrosine produce cleavage at the F-L bond, placing these residues in the P4' position. These data indicate that insulysin contains an extended active site, with residues in the S2', S3', and S4' subsites contributing to specificity.
|
The effects seen by P2' residues differ from those seen with P2 residues because none of the P2' residues serve as cleavage sites. However, these residues can influence the cleavage site. There are a number of possible explanations for this, the simplest being that lysine is a poor P1 residue. The rate enhancements produced by P2' residues on cleavage at the R-K bond probably result from increased binding energy produced through distal subsite interactions. Those residues that produce additional cleavage sites probably destabilize interactions at the S2 subsite and/or produce favorable interactions at the S3 subsite.
The data in Tables II and III also provide insight into the rate-determining step of the reaction. Because the insulysin reaction involves the conversion of a single substrate into two products, there are three potential rate-determining steps. These are the release of the N-terminal fragment of the substrate, the release of the C-terminal fragment of the substrate, or cleavage of the peptide bond. As seen in Table II, with Abz-GGFXRKVGQ-EDDnp as substrate, variable amino acids in the N-terminal portion of the substrate produce a greater than 7-fold variation in kcat when one considers formation of the common C-terminal product RKVGQ-EDDnp. Similarly, a 14-fold variation for formation of the common C-terminal product KVGQ-EDDnp is seen. If release of these C-terminal fragments were rate-limiting, these products would all exhibit the same kcat. Similarly, the data in Table III show a greater than 9-fold variation for release of the common N-terminal product Abz-GGFLR when the amino acid in the C-terminal part of the substrate is varied. These data indicate that release of the N-terminal fragment is also not the rate-limiting step. One is left to conclude that the cleavage of the peptide bond is the rate-limiting step of the reaction, at least with the peptides used in this study.
Together, the data support our previous proposal (13) that insulysin
prefers to cleave at hydrophobic (preferably aromatic hydrophobic)
residues and basic residues. The data further suggest that the
specificity of insulysin is directed toward the amino side of these
residues. Multiple subsite interactions are possible, permitting
different alignments of the substrate relative to the catalytic site.
These interactions determine the site of bond cleavage and to a lesser
extent the rate of bond cleavage. Together these factors provide
insights into the complex cleavage pattern observed when insulysin acts
on physiological substrates.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Richard Roth of Stanford University for providing us with the cDNA clone to rat insulysin.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institute on Drug Abuse Grants DA02243 and DA 07062.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry, College of Medicine, Chandler Medical Center, University of Kentucky, 800 Rose St., Lexington, KY 40536-0084. Tel.:
859-323-5549; Fax: 859-323-1727; E-mail: lhersh@pop.uky.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M008702200
2 The nomenclature of Schechter and Berger (26) is used in which the residue on the carboxyl side of the bond being cleaved is designated as the P1 residue, and the residue on the amino side of the bond being cleaved is designated as the P1' residue. Subsequent residues are designated P2', P3', P2, P3, etc. With the fluorogenic peptides used in this study, a number of them are cleaved at multiple positions. For simplicity and based on the parent peptide Abz-GGFLRKVGQ-EDDnp, which is cleaved at the R-K bond, we refer to the R residue as the P1 residue and the K residue as the P1' residue.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HPLC, high performance liquid chromatography; Abz, 2-aminobenzyl; EDDnp, ethylenediamine-2,4-dinitrophenyl.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Mirsky, I. A., and Broh-Kahn, R. H. (1949) Arch. Biochem. 20, 1-9 |
2. |
Duckworth, W. C.,
Bennett, R. G.,
and Hamel, F. G.
(1998)
Endocr. Rev.
19,
608-624 |
3. | Seta, K. A., and Roth, R. A. (1997) Biochem. Biophys. Res. Commun. 231, 167-171[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Vekrellis, K.,
Ye, Z.,
Qiu, W. Q.,
Walsh, D.,
Hartley, D.,
Chesneau, V.,
Rosner, M. R.,
and Selkoe, D. J.
(2000)
J. Neurosci.
20,
1657-1665 |
5. |
Qiu, W. Q.,
Walsh, D. M.,
Ye, Z.,
Vekrellis, K.,
Zhang, J.,
Podlisny, M. B.,
Rosner, M. R.,
Safavi, A.,
Hersh, L. B.,
and Selkoe, D. J.
(1998)
J. Biol. Chem.
273,
32730-32738 |
6. | Authier, F., Posner, B. I., and Bergeron, J. J. M. (1996) Clin. Invest. Med. 19, 149-160[Medline] [Order article via Infotrieve] |
7. | Roth, R. A., Mesirow, M. L., Yokono, K., and Baba, S. (1984) Endocr. Res. 10, 101-112[Medline] [Order article via Infotrieve] |
8. | Muller, D., Schulze, C., Baumeister, H., Buck, F., and Richter, D. (1992) Biochemistry 31, 11138-11143[Medline] [Order article via Infotrieve] |
9. | Garcia, J. V., Gehm, B. D., and Rosner, M. R. (1989) J. Cell Biol. 109, 1301-1307[Abstract] |
10. |
Semple, J. W.,
Ellis, J.,
and Delovitch, T. L.
(1989)
J. Immunol.
142,
4184-4193 |
11. |
Duckworth, W. C.,
Robert, G. B.,
and Hamel, F. G.
(1994)
J. Biol. Chem.
269,
24575-24580 |
12. |
Kupfer, S. R.,
Wilson, E. M.,
and French, F. S.
(1994)
J. Biol. Chem.
269,
20622-20628 |
13. | Safavi, A., Miller, B. C., Cottam, L., and Hersh, L. B. (1996) Biochemistry 35, 14318-14325[CrossRef][Medline] [Order article via Infotrieve] |
14. | Kurochkin, I. V., and Goto, S. (1994) FEBS Lett. 345, 33-37[CrossRef][Medline] [Order article via Infotrieve] |
15. | McDermott, J. R., and Gibson, A. M. (1997) Neurochem. Res. 22, 49-56[Medline] [Order article via Infotrieve] |
16. |
Mukherjee, A.,
Song, E.-S.,
Kihiko, M.,
Goodman, J. P., Jr.,
Pyrek, J. S.,
Estus, S.,
and Hersh, L. B.
(2000)
J. Neurosci.
20,
8745-8749 |
17. | Perez, A., Morekki, L., Cresto, J. C., and Castano, E. M. (2000) Neurochem. Res. 25, 247-255[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Perlman, R. K.,
Gehm, B. D.,
Kuo, W.-L.,
and Rosner, M.
(1993)
J. Biol. Chem.
268,
21538-21544 |
19. |
Perlman, R. K.,
and Rosner, M.
(1994)
J. Biol. Chem.
269,
33140-33145 |
20. |
Gehm, B. D.,
Kuo, W.-L.,
Perlman, R. K.,
and Rosner, M.
(1993)
J. Biol. Chem.
268,
7943-7948 |
21. | Baumeister, H., Muller, D., Rehbein, M., and Richter, D. (1993) FEBS Lett. 317, 350-354 |
22. | Csuhai, E., Juliano, M. A., Pyrek, J. S., Harms, A. C., Juliano, L., and Hersh, L. B. (1999) Anal. Biochem. 269, 149-154[CrossRef][Medline] [Order article via Infotrieve] |
23. | Cleland, W. W. (1979) Methods Enzymol. 63, 103-137[Medline] [Order article via Infotrieve] |
24. | Chagas, J. R., Juliano, L., and Prado, E. (1991) Anal. Biochem. 192, 419-425[Medline] [Order article via Infotrieve] |
25. | Gershkovich, A. A., and Kholodovych, V. V. (1996) J. Biochem. Biophys. Methods 33, 135-162[CrossRef][Medline] [Order article via Infotrieve] |
26. | Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162[Medline] [Order article via Infotrieve] |