(Received for publication, September 20, 1996, and in revised form, November 6, 1996)
From the Departments of Biological Structure and
¶ Ophthalmology, University of Washington,
Seattle, Washington 98195-7420 and § Department of
Molecular Biology, University of Nijmegen, Toernooiveld,
Nijmegen 6525 ED, The Netherlands
The polymerase chain reaction was used to amplify
a cDNA sequence encoding the human B-crystallin. The amplified
cDNA fragment was cloned into the bacterial expression vector
pMAL-c2 and expressed as a soluble fusion protein coupled to
maltose-binding protein (MBP). After maltose affinity chromatography
and cleavage from MBP by Factor Xa, the recombinant human
B-crystallin was separated from MBP and Factor Xa by anion exchange
chromatography. Recombinant
B-crystallin was characterized by
SDS-polyacrylamide electrophoresis (PAGE), Western immunoblot analysis,
Edman degradation, circular dichroism spectroscopy, and size exclusion
chromatography. The purified crystallin migrated on SDS-PAGE to an
apparent molecular weight (Mr ~22,000) that
corresponded to total native human
-crystallin and was recognized on
Western immunoblots by antiserum raised against human
B-crystallin
purified from lens homogenates. Chemical sequencing, circular dichroism
spectroscopy, and size exclusion chromatography demonstrated that the
recombinant crystallin had properties similar or identical to its
native counterpart. Both recombinant
B-crystallin and MBP-
B
fusion protein associated to form high molecular weight complexes that
displayed chaperone-like function by inhibiting the aggregation of
alcohol dehydrogenase at 37 °C and demonstrated the importance of
the C-terminal domain of
B-crystallin for chaperone-like
activity.
The cytoplasm of human lens cells is composed predominantly of a
group of soluble globular proteins known as the crystallins. The
crystallin proteins in the human lens are categorized as the - and
/
-crystallin families (1). The structure, stability, and short
range order of the crystallins are thought to contribute to the
transparency of the vertebrate ocular lens (2).
-Crystallin is the
most abundant of the crystallins in the vertebrate lens and is composed
of two Mr 20,000 subunits,
A and
B, which
associate to form high molecular weight oligomers ranging from
approximately 3 × 105 to 1.2 × 106
daltons (3). The two proteins are derived from single copy genes that
share approximately 55% identity (1). The
-crystallins also share
sequence similarity with the small heat shock proteins of numerous
species (4). The tertiary structures of the
-crystallins have not
been elucidated, but it is possible that they share the two-domain
structure found in other crystallins (5, 6). In vitro a
chaperone-like activity has been described for bovine
-crystallins
in suppressing the aggregation of proteins denatured at high
temperature (7-13). Human
B-crystallin expression is found under
normal conditions in many nonlens cells and tissues, including heart,
brain, skeletal muscle, kidney, placenta, and lung (14-22) and, like
the ubiquitous small heat shock proteins, is dramatically up-regulated
in response to stress and under pathological conditions (23-30).
B-Crystallin has biochemical properties that result in its
copurification with mammalian heat shock protein 28 from human skeletal
muscle (18). The
B-crystallin gene has been shown to contain a heat
shock element in its promoter that may be subject to a heat-regulated
control mechanism (31).
The chemical nature of the interactions between human -crystallins
and other proteins is poorly understood because of the difficulty with
isolation of sufficient quantities of unmodified protein from human
lenses. For this reason, recombinant techniques have been used to
characterize structure-function relationships of the individual
-crystallin subunits. The conformational properties of substrate
proteins bound to recombinant human
A-crystallin was recently
reported (32); however, unlike human
B-crystallin, human
A-crystallin lacks a heat shock element in its promoter, is not
induced by stress or pathological conditions, and has a limited
expression pattern in the body. In this article we report expression
and characterization of the small heat shock protein human
B-crystallin cloned from a fetal lens cDNA library. To the best
of our knowledge this is the first demonstration that human
B-crystallin displays molecular chaperone activity. The recombinant
expression system described here provided an excellent source of
unmodified human
B-crystallin that assembled into a high molecular
weight oligomer, and displayed chaperone-like activity against protein
aggregation.
RNA was
isolated from 9-12-week human fetal lenses as described previously
(33). Oligo(dT)-primed cDNA was prepared from 2 µg of total RNA
using the Boehringer Mannheim cDNA synthesis kit according to the
manufacturer's instructions. The cDNA was EcoRI-linkered, digested with EcoRI, and ligated
to EcoRI gt11 arms (Promega, Leiden, The Netherlands).
After in vitro packaging, the phages were plated on
Escherichia coli Y1090 cells.
The
coding region for human B-crystallin was isolated from the fetal
lens cDNA library by polymerase chain reaction and inserted into
the cloning vector pCRTMII (Invitrogen, San Diego, CA). The
following primers, which correspond to the 5
- and 3
-ends of the
coding region for human
B-crystallin (16, 36), were synthesized and
used in the polymerase chain reactions:
5
-CCAGAATTCATGGACATCGCCATCCACCAC-3
(forward) and 5
-CCATCTAGATCATTTCTTGGGGGCTGCGGT-3
(reverse). EcoRI and
XbaI sites were attached to the 5
-ends of the forward and
reverse primers, respectively. The coding region for the human
B-crystallin was then removed from the pCRTMII vector at
its flanking restriction sites and ligated into
EcoRI-XbaI-digested pMALTM-C2 (New
England Biolabs, Beverly, MA) to produce pMAL-C2-
B3. The coding
region was inserted downstream of the malE gene of E. coli, which encodes MBP,1 resulting in
the expression of a MBP-
B fusion protein. The coding region of this
expression construct was confirmed by DNA sequence analysis using the
dideoxy chain termination method (34).
The pMAL-C2-B3 expression plasmid was used to
transform competent E. coli JM109 cells (Stratagene, San
Diego, CA). One liter of L broth that contained 100 µg/ml ampicillin
was inoculated with 10 ml of an overnight culture of the transformed
E. coli cells, and cells were grown with vigorous shaking at
37 °C. The cells were incubated until the culture reached an optical
density of ~0.5 at A = 600 nm, at which point protein
expression was induced by addition of
isopropyl-
-D-thiogalactopyranoside to a final concentration of 0.3 mM (Sigma). Four
hours after induction, cells were harvested by sedimentation and
resuspended in 50 ml of column buffer (20 mM Tris-HCl, pH
7.4, 200 mM NaCl, and 1 mM EDTA). Cells were
stored overnight at
20 °C. After thawing, the cells were disrupted
by sonication on ice by eight 30-s cycles at 70 watts on a Branson
(Plainview, NY) Ultrasonics power sonifier. Insoluble cellular debris
was removed by sedimentation at 9,000 × g for 30 min
at 4 °C. Soluble fusion protein present in the supernatant was
purified by adsorption to a 10-ml amylose resin affinity column (New
England Biolabs) for 1 h at 25 °C. After washing with 10 column
volumes of column buffer, bound fusion protein was eluted using column
buffer that contained 10 mM maltose. Preparations of fusion
protein were concentrated using Centriplus-10 microconcentrators (Amicon, Beverly, MA). Preparations of fusion protein contained <5%
contaminating proteins as assessed by SDS-PAGE and Coomassie Blue
staining. Concentrations of purified fusion protein were determined by
a protein assay (Bio-Rad).
MBP was
cleaved from human B using the protease Factor Xa, the recognition
sequence of which is encoded in the pMAL-c2-
B3 vector within the
fourth and fifth codons 5
from the coding region for
B-crystallin.
Recombinant human
B-crystallin was purified by anion exchange
chromatography in the presence of 8 M urea. Briefly,
cleaved fusion protein was dialyzed against ion exchange buffer (20 mM Tris-HCl, pH 8.0, 25 mM NaCl, 1 mM EDTA, and 8 M urea). MBP and Factor Xa were
separated from human
B-crystallin by absorption to a 10-ml column of
Q-Sepharose anion exchange resin (Pharmacia Biotech Inc.). In the
presence of ion exchange buffer, recombinant human
B-crystallin had
no affinity for the Q-Sepharose anion exchange resin and hence could
easily be separated from MBP and Factor Xa by this method. Preparations
of recombinant human
B-crystallin were dialyzed overnight against
ion exchange buffer that lacked 8 M urea to promote
reoligomerization and then concentrated on Centriplus-10
microconcentrators. Preparations of recombinant human
B-crystallin
contained <5% contaminating proteins as assessed by SDS-PAGE and
Coomassie Blue staining. Concentrations of purified recombinant human
B-crystallin were determined by a protein assay (Bio-Rad).
Proteins were analyzed on 4-20% polyacrylamide
electrophoretic gels in the presence of 0.1% SDS (Novex, San Diego,
CA) and were stained with Coomassie Blue R-350 (Pharmacia). Proteins
were electrophoretically transferred to a polyvinylidene difluoride membrane using a Blot Module II (Novex). Antiserum raised against B-crystallin purified directly from human lens homogenates was used
as a primary antibody (35). For immunodetection, alkaline phosphatase
conjugated to goat anti-rabbit IgG antibody and
5-bromo-4-chloro-3
indolylphosphate and p-nitro blue
tetrazolium chloride (Bio-Rad) were used.
N-terminal
sequencing of the purified human B-crystallin was performed by
sequential Edman degradation (15 cycles) using an Applied Biosystems
477A liquid phase protein sequencer with an on-line 120A
phenylthiohydantoin analyzer after immobilization on a polyvinylidene
difluoride membrane.
Circular dichroism spectra were obtained using a Jasco 720 spectropolarimeter. A 0.5-mm path length cell was used. Sixteen scans were averaged per sample, and spectra of the buffers were subtracted from the spectra of the protein samples.
Reassociation of Human MBP-Recombinant human MBP-B and
B-crystallin
were fractionated by size exclusion chromatography on a Macrosphere GPC
300-Å, 7 µm, 250 × 4.6-mm size exclusion column (Alltech,
Deerfield, IL) using a Hewlett-Packard (Palo Alto, CA) 1090 high
performance liquid chromatography with the following mobile phase: 0.1 M KH2PO4 (pH 7.0) and 0.2 M NaCl, at a flow rate of 0.1 ml min
1. High
molecular weight protein standards (Pharmacia) were used to calibrate
the column.
The effect of human
B-crystallin on protein aggregation was measured as described
previously (7). Briefly, the aggregation of ADH at 37 °C was
measured as the apparent optical density at A = 360 nm
using a Shimadzu (Kyoto, Japan) UV-160 UV-visible recording spectrophotometer equipped with a temperature-controlled cuvette holder. In a total reaction volume of 400 µl, 5 µM
equine liver ADH (Sigma) was incubated at 37 °C
with varying amounts of purified human MBP-
B or
B-crystallin. All
reagents were diluted into the following reaction buffer: 50 mM sodium phosphate buffer (pH 7.0), 0.1 M
NaCl, and 2 mM EDTA. Stock solutions were stored on ice
until they were mixed at room temperature and quickly placed in the
temperature-controlled sample chamber of the spectrophotometer. The
temperature in the cuvette was monitored using a bead thermistor installed in a cuvette within the sample chamber. The optical density
in each cell was recorded every 60 s.
The
cDNA-coding region of B-crystallin, isolated by polymerase chain
reaction from a human fetal lens cDNA library, was ligated into the
plasmid pMALTM-c2 to produce pMAL-c2-
B3. Double-stranded
dideoxy sequencing of both strands encoding pMAL-c2-
B3 demonstrated
that the
B sequence was 100% identical to the corresponding exon
sequence of the
B-crystallin gene and to the coding regions of
partial- and full-length
B-crystallin cDNA clones (16, 36, 37). The N terminus of the recombinant human
B-crystallin contained 4 additional amino acids: isoleucine, serine, glutamic acid, and phenylalanine, all of which are derived from the insertion of the human
B-coding region into the EcoRI and XbaI sites
in the polylinker of pMALTM-c2. Hence, pMAL-c2-
B3
contained an open reading frame of 537 base pairs, predicted to encode
a polypeptide of 179 amino acids after cleavage and separation from
MBP.
Fig. 1 contains SDS-PAGE
(A) and Western immunoblot analysis (B) of the
expression and purification of recombinant human B-crystallin. Fig.
1A is a Coomassie Blue stain of a polyacrylamide
electrophoretic gel run in the presence of 0.1% SDS. Control of
induction of MBP-
B fusion protein expression was apparent in crude
cell lysates of bacterial cultures transformed with the human
B-crystallin expression construct before and after the addition of
isopropyl-
-D-thiogalactopyranoside (Fig. 1A, lanes
1 and 2, respectively). Treatment of the
affinity-purified fusion protein (Fig. 1A, lane 3) with the
serine protease Factor Xa demonstrated that this fusion protein was
cleaved into two distinct polypeptides (Fig. 1A, lane 4)
that migrate to molecular weights corresponding to native MBP
(~43,000) and native human
B-crystallin (~22,000). The
recombinant human
B-crystallin was isolated by anion exchange
chromatography in the presence of 8 M urea and was found to
contain <5% contaminating proteins as assessed by SDS-PAGE (Fig.
1A, lane 5). Further confirmation of the expression and
purification of recombinant human
B-crystallin was demonstrated by
Western immunoblot analysis using anti-human
B-crystallin antiserum
(Fig. 1B). One predominant immunoreactive band was observed
in the purified uncleaved fusion protein (Fig. 1B, lane 1),
in the mixture of cleaved fusion protein (Fig. 1B, lane 2),
and with purified human
B-crystallin (Fig. 1B, lane 3).
Chemical Sequencing Results of Human
To
verify that the Mr ~22,000 band corresponded
to human B-crystallin, the cleaved fusion protein mixture was
immobilized on a polyvinylidene difluoride membrane, and the
Mr ~22,000 band was subjected to sequential
Edman degradation for 15 cycles. The chemical sequencing results
demonstrated that after the first four vector-derived N-terminal amino
acids (ISEF) the next 11 residues of this protein (MDIAIHHPWIR) were
100% identical to the deduced amino acid sequence predicted from the
exon sequence of the
B gene (36).
In Fig.
2 the far UV circular dichroism spectra of recombinant
human B-crystallin in the absence and presence of 1% SDS are presented. The spectrum of recombinant human
B-crystallin in the
presence of buffer alone (Fig. 2, solid line) resembles
published spectra for total native bovine
-crystallin as well as
recombinant and native bovine
A-crystallin and is typical of a
spectrum for a predominantly
-pleated structure (12, 38-40). On
addition of SDS to a final concentration of 1%, an increase in molar
ellipticity was observed at all wavelengths, indicative of major
structural changes (Fig. 2, dashed line) in the spectrum, as
reported previously for total native bovine
-crystallin (38).
Reassociation of Human MBP-
Fractionation of the recombinant MBP-B fusion
protein and the
B-crystallin alone by chromatography on a size
exclusion column under nondenaturing conditions demonstrated that they
formed high molecular weight oligomers between 2.32 × 105 and 4.40 × 105 in size (Fig.
3).
Complete Inhibition of the Thermally Induced Aggregation of ADH by Human MBP-
The apparent optical density
was a direct measure of the aggregation of ADH at 37 °C over 60 min.
Human B-crystallin and MBP-
B suppressed the aggregation of ADH
over a range of concentrations from 1 to 100 nM (Fig.
4, A and B, respectively). We
assumed molecular weights of 8.0 × 104 for ADH,
1.3 × 106 for MBP-
B-crystallin, and 4.4 × 105 for
B-crystallin. At a molar ratio of 200:1
(ADH:human MBP-
B/
B-crystallin), partial inhibition of ADH
aggregation was observed. At a molar ratio of 50:1 (ADH:human
MBP-
B/
B-crystallin), complete suppression of ADH aggregation was
observed. At a molar ratio of 5,000:1 (ADH:human MBP-
B/
B-crystallin), no effect on the aggregation of ADH was observed. As a control, MBP alone was tested at a molar ratio of 1:1 or
lower (ADH:MBP), and no effect on the aggregation of ADH was observed
(data not shown).
Human lens crystallins undergo extensive posttranslational
modifications during the aging process (41-44). These modifications lead to protein heterogeneity, which has precluded the successful purification of large amounts of homogenous human crystallins to be
used for biophysical, functional, and structural analyses. Here we
report for the first time expression and characterization of functional
human B-crystallin in E. coli.
DNA primers designed against the 5- and 3
-ends of the
B coding
region successfully amplified a ~525-base pair cDNA from a human
fetal lens cDNA library, confirming that this gene is transcriptionally active in the human lens (data not shown). The amplified coding region for this crystallin was subcloned into a
bacterial expression plasmid and expressed in the bacterial cytoplasm
as a soluble fusion protein coupled to MBP. After cleavage and
separation from MBP, SDS-PAGE and Western immunoblot analysis confirmed
that the recombinant protein migrated to its predicted molecular weight
(Mr ~22,000). This polypeptide was recognized by antiserum raised against human
B-crystallin purified directly from lens homogenates. N-terminal chemical sequencing demonstrated that
after the first four vector-derived amino acids (ISEF) the recombinant
human
B-crystallin was 100% identical to the deduced amino acid
sequence predicted from the exon sequence of the human
B-crystallin
gene (36). Although the N-terminal methionine of the recombinant
B-crystallin is preceded by four additional residues (isoleucine,
serine, glutamic acid, and phenylalanine), circular dichroism
spectroscopic analysis indicated that the secondary structure of the
reassociated
B resembled the secondary structures previously
reported for total purified bovine
-crystallin as well as
recombinant and purified bovine
A-crystallin (12, 38-40).
On a size exclusion chromatography column recombinant human MBP-B
and
B-crystallin eluted as high molecular weight oligomers similar
in size (between 2.3 × 105 and 4.4 × 105) to total
-crystallin purified directly from human
eye lenses (45). Interestingly, MBP-
B fusion protein was able to
associate into a high molecular weight oligomer despite the presence of the Mr 42,700 N-terminal fusion partner MBP.
Assuming molecular weights of 1.3 × 106 for MBP-
B
and 4.4 × 105 for
B-crystallin, both the human
MBP-
B and
B-crystallin displayed molecular chaperone activity, as
demonstrated by their complete suppression of the aggregation of ADH at
a molar ratio of approximately 50:1 (ADH:crystallin). When the
molecular weights of the subunits of MBP-
B and
B-crystallin were
used (6.5 × 104 and 2.2 × 104,
respectively), complete suppression of the aggregation of ADH was
observed at a molar ratio of approximately 2.5:1 (ADH:crystallin). A
systematic evaluation of the precise stoichiometry involved in the
suppression of ADH aggregation by MBP-
B and
B-crystallin will
need to be addressed in the future.
It was recently demonstrated using site-directed mutagenesis that
specific residues at the C terminus of recombinant bovine A-crystallin influenced its reassociation and chaperone activity (46). To date, no mutants of human
B-crystallin have been
characterized; however, in the studies presented here the presence of
four extra N-terminal residues had no observable effect on the
secondary structure, reassociation, and chaperone activity of
recombinant human
B-crystallin. Strikingly, human MBP-
B formed a
large functional structure and was able to completely suppress the
aggregation of ADH at concentrations identical to those used with
recombinant human
B-crystallin alone. Taken together, these results
contribute to the evidence that the well conserved C-terminal domain of
the
-crystallins is responsible for the proper assembly into large functional oligomers (5, 6) .
At the amino acid level B-crystallin is remarkably well conserved
among mammalian species. This high degree of conservation among species
may indicate a critical function for
B-crystallin in nonlens cells,
in which expression occurs in response to environmental and
pathological stress. The successful expression of functional recombinant human
B-crystallin creates the first opportunity to
characterize the chemical basis of the interactions between
B and
other proteins in lens and nonlens cells under normal and pathological
conditions.
We thank Dr. Larry Takemoto for the kind gift
of anti-human B-crystallin antiserum and Chris Ganders, Dr. Hiro
Matsushima, Alireza Milaninia, and Melissa Valdez for technical
support. DNA sequencing reactions were performed by the Seattle
Biomedical Research Institute, and chemical protein sequencing was
performed by the Molecular Pharmacology Facility at the University of
Washington.