(Received for publication, September 12, 1995; and in revised form, December 13, 1995)
From the
The sequence of human B1-crystallin cDNA encoded a protein
of 251 amino acids in length. Mass spectrometric analysis of intact
B1 from young human lens confirmed the deduced amino acid
sequence. Lenses of human donors newborn to 27 years of age also
contained partially degraded forms of
B1 missing 15, 33, 34, 35,
36, 39, 40, and 41 amino acid residues from their N-terminal
extensions. The similarity of the cleavage site between residues 15 and
16 in human
B1 to the cleavage occurring in bovine
B1
suggested that lenses of both species may contain a similar proteolytic
activity. The remaining cleavage sites occurring in human
B1 did
not closely match those occurring in other species, possibly due to the
widely divergent amino acid sequence of the N-terminal extension of
B1 amoung species. Results from animal models suggest that
cleavage of the N-terminal extension of
B1-crystallin could
enhance protein insolubilization and cataract in lens. However, the
presence of partially degraded
B1-crystallins in both
water-soluble and water-insoluble fractions of lenses of young donors
suggested that the rate that proteolyzed
B1-crystallins become
water-insoluble is relatively slow in humans.
The lens is a unique organ in that 20-60% of its wet
weight is composed of proteins called crystallins(1) .
Mammalian crystallins can be divided into two broad classes, the
-crystallins, which share homology to heat shock proteins and
exhibit chaperone-like properties (2) , and the
/
-crystallin family, which like
-crystallins, also share
a common ancestor but perform primarily a structural role. The
individual proteins of
/
-crystallin family contain four
homologous motifs, each folded in a ``Greek key''
pattern(3) . These motifs are organized into two equivalent
domains, which are connected by approximately 4 amino acid residues,
the entire structure thus forming the core of the protein. There are
seven different
-crystallin proteins in bovine lens. These are
named
B1,
B2,
B3,
A1,
A2,
A3, and
A4(4) . Each crystallin transcript is translated to yield
a single protein species, except the mRNA coding for
A3, which
also yields
A1, owing to an alternate downstream initiation
codon(5) . Orthologous
-crystallins are also found in
other vertebrate species. Except for
A2, orthologs of all known
-crystallin proteins have been demonstrated in rat(6) .
While the presence of all protein products has not been established in
the chicken, the cDNAs of all six
-crystallin transcripts have
been sequenced(7) . In human lens, the presence of
B1(8) ,
B2(9) ,
A3(10) , and
A4 (
)have been documented. However, to date, there is
no evidence for the significant accumulation of
A1,
A2, or
B3 proteins in postnatal human lens.
All -crystallins
contain N-terminal extensions ranging from 10 to 58 amino acids in
length as well C-terminal extensions of approximately 15 amino acids in
the
B-crystallin subgroup(4) . These N- and C-terminal
extensions are missing in
-crystallins. Since
-crystallins
form complex mixtures of homodimers, heterodimers, and higher order
structures, while
-crystallins do not, it has been hypothesized
that the extensions function to stabilize the intermolecular
associations between
-crystallins(11) . It is likely not a
coincidence that
B1, which contains the longest N-terminal
extension, is also selectively found in the largest
-crystallin
aggregate(12) . Thus, recent work has explored the possible
function of
-crystallin N-terminal extensions in stabilizing the
structure of
-crystallin
oligomers(13, 14, 16, 17) . We
hypothesize that the presence of
-crystallin N-terminal extensions
is important, because removal of from 2 to 49 residues from the
N-terminal extensions of various
-crystallin subunits in lenses of
young rodents was associated with protein insolubilization and cataract
formation(6, 18) . The mechanism of protein
insolubilization following shortening of the N-terminal extensions is
unknown. However, it presumably involves an alteration in the manner
the
-crystallin subunits oligomerize. The specific cleavages in
the N-terminal extensions may result from activation of the protease
calpain II(18) . Similar cleavage of
-crystallin
N-terminal extensions and insolubilization of
-crystallins also
occurs during maturation of normal rat lens(19) . However,
these older lenses may remain transparent due to the relatively slower
rate that the process ensues during normal development.
A major
focus of these laboratories is to determine if similar partial
degradation and insolubilization of -crystallins also occurs in
human lenses. In human lenses, the amount of water-soluble
-crystallin decreases dramatically with aging, and partial
degradation of
B2 has been reported(20) . Similarly, a
29-kDa
-crystallin identified as
B1, was prominent in fetal
human lenses, but it was nearly gone by age 5(21) . Thus,
evidence exists for the partial degradation of
-crystallins during
aging of human lenses. However, few detailed studies exist that examine
cleavage sites in human
-crystallins. These studies are limited by
the lack of sequence data for human
-crystallins. Only the
complete sequences of human
B2 and
A3 are
known(22, 23) . This study reports the sequence of
human
B1 cDNA and confirms the deduced amino acid sequence by mass
spectrometry. With the knowledge of the sequence, we then found that
partially degraded forms of
B1 missing various portions of its
N-terminal extension were present in both water-soluble and
water-insoluble fractions of human lens.
Figure 1:
The nucleotide
sequence of human B1-crystallin cDNA. The
B1 cDNA was
amplified by 5`- and 3`-RACE PCR, cloned, and both strands were
sequenced. The numbers to the right indicate the nucleotide
numbers, while the numbers below indicate the amino acid
number of the deduced protein sequence of 251 residues. The poly(A)
addition signal is underlined.
There are evolutionary constraints upon the sequence of
the core protein of -crystallin subunits that are required for
proper folding of the four Greek key motifs, and maintenance of proper
interactions at the subunit interfaces of
-crystallin
dimers(25, 30, 31) . Because of this, amino
acid residues 58-234, which contained motifs 1-4 and the
connecting peptide of human
B1, exhibited 89, 88, and 77% sequence
identity, respectively, with the reported sequences of orthologous rat,
bovine, and chicken
B1(25, 26, 32) . As
previously noted during comparison of rat, bovine, and chicken
B1
sequences (30) , the N-terminal extension of human
B1
exhibited the greatest divergence in sequence between species (Fig. 2). The human
B1 N-terminal extension exhibited only
61, 56, and 18% sequence identity with bovine, rat, and chicken
B1
N-terminal extensions, respectively. Most notable was the loss of the
Pro-Ala repeats found between residues 35 and 44 in bovine
B1, and
the poor conservation of the Pro-X repeats found between
residues 29-38 in rat
B1 (Fig. 2). Similarly, the
bovine, rat, and chicken
B1 C-terminal extensions shared only 78,
72, and 33% sequence identity with the human
B1 C-terminal
extension, respectively.
Figure 2:
Alignment of the N-terminal extensions of
human, bovine(26) , rat(25) , and chicken
B1(32) . Gaps were introduced at dashed regions to allow maximal alignment of
B1 sequences. Arrows above each sequence show known cleavage sites occurring in
vivo within the N-terminal extension of each
species(19, 34) . No cleavage sites have been reported
for chick
B1. The cleavage sites in human
B1 resulting in the
removal of 15, 33, 34, 35, 36, 39, 40, and 41 residues were determined
by detection of proteins and/or tryptic peptides with masses matching
the predicted masses of the indicated cleavage products. Cleavage sites
resulting in the removal of 15, 40, and 41 residues from human
B1
were confirmed by either direct Edman sequencing or MS/MS
analysis.
Figure 3:
Coomassie Blue-stained two-dimensional
electrophoretic gel containing total soluble protein from a single pair
of newborn human lens. The identities of the major -,
-, and
-crystallin species are indicated. The parent
B1 protein, and
its partial degradation product,
B1(-15), were cut from
these blots and trypsinized, and the masses of the resulting fragments
were measured as shown in Fig. 4.
Figure 4:
Masses of peptides found in the tryptic
digests of B1-crystallins. The masses in boldface were found in
the digest of the intact
B1 from a newborn lens, confiming the
deduced amino acid sequence of
B1 shown in Fig. 1. a, the 613-Da tryptic peptide was found in the digest of the
protein marked
B1(-15) in Fig. 3, thus confirming the
cleavage site in this protein between residues 15 and 16. b,
masses of 2 out of 7 other tryptic fragments, indicating further
cleavage of
B1 during maturation. The identities of these tryptic
fragments and others with asterisks were confirmed by other
experiments discussed in the text.
The regions containing intact B1 and
B1(-15) were digested with trypsin, and the masses of the
resulting fragments were analyzed. The peptides from these digests gave
masses corresponding to all expected tryptic peptides in the mass range
analyzed, confirming the deduced amino acid sequence (Fig. 4).
The mass of 545 Da for the N-terminal peptide indicated that the
N-terminal methionine was removed, and that the N-terminal Ser was
acetylated. The mass of the peptide containing Cys-79 appeared 71 Da
higher than expected due to the formation of an acrylamide adduct
during the two-dimensional gel separation(33) . The deduced
protein sequence in Fig. 1has a calculated molecular mass of
27,935 Da, agreeing very well with the ESIMS-determined molecular mass
of 27,933 Da reported by He et al.(10) . Mass spectral
analysis of
B1(-15) also gave all the expected tryptic
peptides, except those corresponding to residues 1-5 and
6-21. The presence of a peptide with a molecular mass
corresponding to peptide 16-21 (613 Da) confirmed the Edman
sequencing data, indicating that the protein had been cleaved between
Asn-15 and Pro-16 (Fig. 4). A peptide with a mass corresponding
to the C-terminal tryptic fragment indicated the presence of an intact
C terminus in the young lens.
Figure 5:
Electrospray ionization mass spectrum of a
fraction from the water-soluble -crystallins of the 20-year-old
lens. The three peaks have masses that fit degradation
products 40-251 (24,391 Da), 35-251 (24,834 Da), and
16-251 (26,535 Da). The other peaks have not been
identified.
Mass spectral analysis of the tryptic peptides
produced from the water-soluble lens crystallins of young adult (age
27) suggested that additional forms of partially degraded
B1-crystallin were present. Even though this digest was a mixture
of peptides from several proteins, peptides encompassing over 75% of
B1-crystallin could be identified from comparisons of their
molecular masses and HPLC elution times with those found in the digest
of
B1 from the newborn lens. Expected peptides derived from the N
terminus of intact
B1 and
B1 (16-251) were not evident,
probably because the N-terminal peptides from these species were
present at very low levels, and the mass spectral response was masked
by the presence of other more responsive peptides. However, the ESIMS
data summarized above indicated that cleavage at Lys-49 by trypsin
should produce peptides corresponding to peptides 35-49,
40-49, and 41-49. Ions with molecular masses corresponding
to these three peptides were found. In addition, the digest contained
ions with molecular masses consistent with the presence of peptides
34-49, 36-49, 37-49, and 42-49, indicating that
there are numerous other N-terminal degradation sites of
B1-crystallin evident by age 27.
Because the tryptic digests of water-insoluble proteins were a complex mixture, the identities of many of the peptides, particularly those indicating the presence of N-terminal degradation, were confirmed. One of three methods was used for confirmation: 1) further digestion with another enzyme, such as pepsin or carboxypeptidase, followed by a second mass spectral analysis of these products; 2) Edman sequencing, if the peptide was pure; or 3) MS/MS analysis of the collision-induced fragmentation pattern of the peptide. Analysis of the MS/MS fragmentation patterns of the peptides at 916 Da and 815 Da, isolated from the 16-year-old lens, showed several fragments that confirmed the identities of the peptides as residues 41-49 and 42-49, respectively. The MS/MS spectrum for peptide 41-49 is shown in Fig. 6.
Figure 6:
MS/MS analysis of MH at
917 Da, confirming its identity as peptide 40-49. The inset shows the expected fragments from the sequence of peptide
40-49 that were found in the
spectrum.
The major finding of this study was that B1-crystallin
of human lens undergoes extensive cleavage at its N-terminal extension
during lens maturation. The first cleavage product of human
B1,
B1 (16-251), was already abundantly evident in lenses of a
newborn donor. This suggested that
B1 may be the most protease
susceptible crystallin in the human lens.
Alcala et al.(21) showed that from 8 months of gestation to 5 years of
age, the percentage of intact B1 in the water-soluble protein of
human lens dropped from 10 to 0.5%. During the same interval, the
percentage of a 27-kDa protein, corresponding to
B1 (16-251)
of the present study, increased from 3.5 to 7%. Thereafter, the 27-kDa
protein decreased, so that by 87 years of age it composed only 1.2% of
the water-soluble lens protein. The loss of intact
B1 and
transient accumulation of the 27-kDa protein also correlated with the
age of the lens fiber. The conversion from intact
B1 to 27-kDa
protein and the subsequent loss of 27-kDa protein was more pronounced
in the deeper lens cortical and nuclear fibers than in the superficial
cortex. These results are consistent with the present findings.
B1
and
B1 (16-251) were most abundant in newborn lenses.
However, by early adulthood, tryptic fragments corresponding to the N
terminus of intact
B1 and
B1 (16-251) were not
detected, while tryptic fragments from forms of
B1 with more
extensively degraded N termini were abundant.
Bovine B1 also
undergoes a similar cleavage as that producing human
B1
(16-251). Bovine
B1 is cleaved between Asn-14 and
Pro-15(34) . The 6 amino acid residues surrounding this
cleavage site are identical between bovine and human
B1 (Fig. 2). This suggested that both species contain a similar
protease capable of cleaving at this site. Rat
B1 does not undergo
cleavage at this site, possible due to the loss of the Pro residue
found at positions 18 and 17 in human and bovine
B1, respectively (Fig. 2). Bovine
B1 also contained a cleavage site between
Ala-11 and Ala-12(34) . Neither mass spectrometric analysis or
Edman sequencing in the present study detected a similar cleavage in
human
B1, possibly due to the lack of sequence identity in this
region between bovine and human
B1.
Further cleavage of human
B1 occurred between residues 33 and 41. ESIMS analysis of
-crystallins from a 2-year-old bovine lens did not
yield molecular masses indicative of similar N-terminal degradation in
this region. Instead, the bovine
B1 from a 2-year-old lens appears
to have undergone even more extensive degradation, since the molecular
masses of the proteins in
-crystallin were all less
than 25,000 Da(35) . Bovine lens
B1 may not have undergone
cleavage in regions similar to human
B1, because residues
35-44 in bovine
B1 contained Pro-Ala repeats not found in
human
B1. Pro-Ala repeats in bovine
B1 and Pro-X repeats in rat
B1 may be more resistant to proteolytic attack
than the corresponding region in human
B1.
Cleavage at five
sites within the N-terminal extension of B1 has also been reported
during maturation of rat lens (Fig. 2). At least two of these
sites in rat
B1 are confirmed calpain II cleavage sites (19) . However, none of the cleavage sites within the
N-terminal extension of rat
B1 corresponded in relative position
to cleavage sites found in human
B1 (Fig. 2). The
difference in the manner rat
B1 and human
B1 are degraded
could be due to a lack of sequence identity at the respective cleavage
sites. Sequences within the N-terminal extensions of
B1 from
various species may have evolved to exhibit specific susceptibilities
to proteolytic attack. Alternatively, the lenses of each species may
contain proteolytic activities with different specificities. The
predominate proteolytic activity responsible for partial degradation of
-crystallin N-terminal extensions in rat lens may be calpain
II(19) . However, the proteases responsible for degradation of
the N-terminal extension of bovine and human
B1 remain unknown.
The extensive cleavage of the N-terminal extension of B1 occurs
quite early in life. This suggested that the cleavage plays an
important role in the maturation process. In rat, the partially
degraded forms of
B1 were found in only the water-insoluble
fraction of the lens(19) . Therefore, the cleavage of the rat
B1 N-terminal extension, as well as the N-terminal extensions of
other
-crystallins, may rapidly induce protein
insolubilization(6) . Such insolubilization may be important in
initiating dehydration and hardening of the rat lens(36) .
However, the results in the present study indicated that human
-crystallins respond quite differently following partial
proteolysis. Partially degraded
B1 was found in both water-soluble
and water-insoluble fractions of young human lens. Also, the majority
of human
B1-crystallin is partially degraded before adulthood, but
most crystallin insolubilization occurs in human lenses after the third
decade of life(15) . Future studies will determine if the
water-insoluble fraction of aged lenses contains a greater proportion
of partially degraded
-crystallins than does the water-soluble
fraction. Also, the relationship between the extent of proteolysis and
lens opacification requires closer examination.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35340[GenBank].