(Received for publication, May 23, 1995; and in revised form, September 26, 1995)
From the
The purification of a 68-kDa hyaluronic acid-binding protein
(HA-binding protein), a homodimer of 34 kDa that binds specifically to
hyaluronic acid, has been reported earlier by us (Gupta, S., Batchu, R.
B., and Datta, K. (1991) Eur. J. Cell Biol. 56, 58-67).
Here, we report the isolation of a partial cDNA clone from a
gt
cDNA expression library of human skin fibroblast by
immunoscreening with HA-binding protein antiserum. The internal
polypeptide sequence (83 residues) of the purified hyaluronic
acid-binding protein is identical to the predicted protein sequence
derived from hyaluronic acid-binding protein cDNA, suggesting the
authenticity of the clone. Interestingly, this hyaluronic acid-binding
protein cDNA sequence has complete homology with the cDNA sequence of a
protein P-32, co-purified with the human pre-mRNA splicing factor SF2
(Krainer, A. R., Mayeda, A., Kozak, D., and Binns, G.(1991) Cell 66, 383-394). Furthermore, the data on the N-terminal
sequence of hyaluronic acid-binding protein and the predicted
polypeptide of P-32 revealed the identical coding sequence of 209 amino
acids for both the proteins. As the identity and functional
characterization of P-32 have not yet been reported, P-32 cDNA was
expressed in Escherichia coli, and the recombinant P-32
protein was purified by hyaluronic acid-Sepharose affinity
chromatography. The recombinant P-32 protein showed
immunocross-reactivity with the polyclonal antibodies raised against
HA-binding protein. The predicted amino acid sequence of the protein
fulfilled the minimal criteria for binding to hyaluronic acid, i.e. two basic amino acids flanking a seven-amino acid stretch, as
reported for other hyaluronic acid-binding proteins. Furthermore, the
hyaluronic acid affinity of the recombinant P-32 protein was confirmed
by biotinylated hyaluronic acid binding assay. The binding of
recombinant P-32 protein to biotinylated hyaluronic acid can be
competed only with excess unlabeled hyaluronic acid, confirming its
specificity toward hyaluronic acid. All these results suggest that both
P-32, co-purified with the human pre-mRNA splicing factor SF2, and
34-kDa hyaluronic acid-binding protein reported by us are the same
protein and that it is a new member of the hyaluronic acid- binding
protein family, the ``hyaladherins.''
Hyaluronic acid (HA), ()a viscoelastic, high
molecular weight, nonsulfated glycosaminoglycan, is ubiquitously
present in both the extracellular space and the nucleus of higher
animals(1, 2, 3, 4, 5) .
The biological functions of HA include not only physiological roles
such as maintenance of matrix structure and water balance (6, 7) but also cellular functions like
proliferation(6, 8) , tissue recognition(7) ,
and locomotion(9) . The cell type-specific functions of HA are
mediated through its interaction with HA-binding proteins(7) .
A number of extracellular as well as cell-surface HA-binding proteins are known to mediate the multifaceted regulations of HA-induced cellular functions(10, 11, 12, 13, 14, 15, 16, 17, 18, 19) . Due to the presence of defined HA-binding motif, these proteins are expected to belong to one family, recently, referred to as ``hyaladherins''(20) . Few of them are molecularly characterized, and their sequence data confirm their distinction.
The purification of 68-kDa homodimer (34-kDa subunit) HA-binding
protein from rat kidney tissue and its specificity toward hyaluronic
acid were confirmed earlier from our laboratory(21) . The
differential expression of this glycoprotein on the cell surface of
AK-5, a transplantable histiocytic tumor cell line, and its role in
cell adhesion were also demonstrated(22) . Ranganathan et
al.(23) observed the in vivo phosphorylation of
this protein in motile spermatozoa and established its role in sperm
maturation, motility, and fertilization. To understand the regulation
of expression of this protein and its precise role in cellular
functions, we have cloned the gene encoding this 34-kDa HA-binding
protein. A gt
human skin fibroblast cDNA expression
library was screened with antibodies raised against this HA-binding
protein. Partial cDNA sequence analysis and the deduced amino acid
sequence match with the amino acid sequence of purified HA-binding
protein. Interestingly, the sequence analysis of the HA-binding protein
confirms its homology with P-32, a protein co-purified with pre-mRNA
splicing factor SF2 from human HeLa cells described by Krainer et
al.(24, 25) . The identity and functional
characterization of P-32 protein was not known earlier. Here, we report
the 34-kDa HA-binding protein to be the same as P-32 from sequence
analysis and also describe the purification of recombinant P-32 protein
by HA-Sepharose column, thus again confirming the identity of P-32
protein as an HA-binding protein. This result is also supported by
immunological and biochemical studies showing the specificity of HA
affinity of this protein.
For reprobing
experiment, proteins were transblotted on ECL Hybond nitrocellulose as
described before. Proteins were probed first with antirecombinant P-32
protein antibodies (1:500), second with goat anti-rabbit horseradish
peroxidase (1:10,000), and bands were visualized by ECL kit by exactly
following the manufacturer's instructions (Amersham). After band
development, the blot was incubated in 62.5 mM Tris-Cl, pH 6.7
containing 100 mM -mercaptoethanol and 2% SDS at 50
°C for 30 min for stripping off the bound antibodies and again
blocked in 10 mM Tris-Cl, pH 8.0, containing 150 mM NaCl (TBS), 0.05% Tween-20 (TBST), and 5% skimmed milk powder
(TBSTS), reprobed with rabbit anti-HA-binding protein antibodies
(1:1000), and then processed by following the previous ECL detection
method described in the manufacturer's kit.
For biotinylated HA transblot experiment, exactly
the same procedure was followed except that the boiled purified
recombinant P-32 protein (with or without 5% -mercaptoethanol in
the sample buffer containing 0.0625 M Tris-Cl, pH 6.8, 2% SDS,
and 10% glycerol) were separated on 12.5% SDS-PAGE and transblotted
onto ECL Hybond nitrocellulose (32) in a buffer containing 25
mM Tris, 192 mM glycine, and 20% methanol, pH 8.3.
Figure 1:
Peptide sequence of
rat kidney 34-kDa HA-binding protein. A, High pressure liquid
chromatogram of HA- binding protein derived peptides. Peptides
D-D
of HA-binding protein are shown. The
CH
CN elution gradient in 0.1% trifluoroacetic acid is shown
by the straight line. B, amino acid sequence of HA-
binding protein peptides separated in panel A. Standard amino
acid code is used. X represents an undetermined amino acid.
Peptide D2 constitutes the N terminus of the HA-binding
protein.
Figure 2: A, triple alignment of cDNA and predicted amino acid sequences of SF2P-32 as published by Honore et al.(35) and Krainer et al.(24) and HA-binding protein in this study. The one-letter amino acid notation is used. The inverted and upright arrows mark the initiation and termination codon, respectively. Potential polyadenylation signals in HA-binding protein cDNA sequence are boxed. The amino acid sequences derived from purified HA-binding protein by peptide sequencing are underlined. Closed triangles indicate potential N-glycosylation sites. The asterisk marks the initiation of mature protein. SF2P32 (H) and SF2P32 (K) denote the sequence of P-32 published by Honore et al.(35) and Krainer et al.(24) , respectively. B, schematic diagram of P-32 sequence published by Honore et al.(35) (a) and Krainer et al.(24) (b) and of HA-binding protein in this study (c). The boxed region in part a represents a proprotein that is post-translationally processed by removal of 73 amino acids at the N terminus (dotted box) to produce a mature protein of 209 amino acids (shaded box). The N terminus of mature HA-binding protein (black region) in part c as determined by peptide sequencing of purified HA-binding protein is missing in the partial cDNA sequence. Diagram is not drawn to scale.
Figure 3: Alignment of amino acid sequence of SF2P-32 or HA-binding protein with that of CD44. The N-terminal region (amino acids 74-161) of mature P-32 or HA-binding protein matched with the transmembrane and cytosolic domain of CD44 (amino acids 273-360) without any break. Box denotes conserved amino acid.
Figure 4: Electrophoretic mobility and immunodetection of recombinant P-32 shown by 12.5% SDS-PAGE (A) of bacterial (BL21(3E)) lysate containing uninduced pET3c plasmid (lane 1, 10 µg), IPTG-induced pET3c plasmid (lane 2, 10 µg), uninduced expression plasmid with P-32 cDNA insert (lane 3, 10 µg), IPTG-induced expression plasmid with P-32 cDNA insert (lane 4, 18 µg), or tissue-derived HA-binding protein (lane 5, 4 µg). Molecular mass markers are included at the right. B, Western blot analysis of a similar gel, electroblotted on nitrocellulose and probed with rabbit anti-HA-binding protein antibodies. Lanes 1-4 contain equal amounts of the same proteins as in panel A. Lane 5 indicates molecular mass markers.
Figure 5: Purification profile and two-dimensional gel electrophoresis analysis of recombinant P-32. A, SDS-PAGE of IPTG-induced bacterial extract containing recombinant P-32 protein (lane 2, 10 µg) and HA-Sepharose affinity-purified recombinant P-32 (lane 3, 5 µg). Low molecular mass markers were loaded in lane 1. B, two-dimensional gel electrophoresis analysis of purified recombinant P-32. HA-Sepharose column eluate (3 µg of protein) was subjected to isoelectric focusing over a pH range of 3.0-10.0. The focused proteins were separated by 10% SDS-PAGE and stained with Coomassie Blue. An arrowhead denotes the P-32 recombinant protein band. Size markers are included on the left.
Figure 6: Immunocross-reactivity between P-32 and HA-binding protein. A, purified HA-binding protein (lane 1, 5 µg) and control purified recombinant P-32 protein (lane 2, 5 µg) were transblotted on nitrocellulose and first detected with the anti-P-32 antibodies. B, after stripping off the anti-P-32 antibodies, the same blot containing HA-binding protein (lane 1) as control was reprobed with anti-HA-binding protein antibodies following the protocol described in the ECL kit.
Figure 7:
A, specific affinity of recombinant P-32
toward HA. Purified HA-binding protein as control (slot 1, 10
µg) and recombinant P-32 (slot 2, 8 µg; slot
3, 6 µg; slot 4, 4 µg; slot 5, 2 µg)
dot-blotted on nitrocellulose was stained with biotinylated HA (1:4000
dilution) that had been previously blocked with milk protein for 2 h (blot D). The other three replicate blots containing the same
amount of the proteins are stained in parallel with the same dilution
of biotinylated HA in presence of 20-fold excess of unlabeled HA (blot C), liver total RNA (blot B), and calf thymus
DNA (blot A). The amount of biotinylated HA bound to the
proteins has been detected using avidin peroxidase (1:1500 dilution)
and the ECL detection system. Only unlabeled HA blocked binding of
HA-binding protein and P-32 to biotinylated HA. Succinic dehydrogenase
has also been used as negative control in the blot, which did not stain
with biotinylated HA (data not shown). B, biotinylated HA
transblot assay: Recombinant purified P-32 (5 µg in each lane) was subjected to SDS-PAGE under reducing
(+-mercaptoethanol, lane 1) and nonreducing
conditions (-
-mercaptoethanol, lane 2),
electroblotted on nitrocellulose, and probed with biotinylated HA as
described under ``Experimental
Procedures.''
We have described here the cloning of the partial cDNA clone for 34-kDa HA-binding protein and confirmed this protein as P-32, a protein that co-purified with pre-mRNA splicing factor SF2 from human HeLa cells(24, 25) . The conclusion is based on the following facts: first, both proteins, namely P-32 and HA-binding protein, are completely identical at cDNA levels; secondly, the predicted amino acid sequence from the cDNA sequence of P-32 protein (24) is again almost identical with partial peptide sequences of purified HA-binding protein; and finally, the identity of P-32 protein as HA-binding protein is confirmed by the presence of known HA-binding motif, purification of recombinant P-32 protein using HA-Sepharose affinity chromatography, the identical electrophoretic mobility, immunocross-reactivity of P-32 protein with anti HA-binding protein antibodies or vice-versa, and the specific affinity of P-32 toward HA.
The functional characteristics of P-32 are not yet known. Krainer et al.(24) reported the sequence of P-32 cDNA, which does not contain any conventional ATG (Met) start codon but initiates with a CTG (Leu) codon. The matching of predicted polypeptide sequence with the N terminus of HeLa cell purified P-32 and the absence of upstream ATG codon or consensus 3` splice sites perhaps had led them to conclude that the mature N terminus does not arise by proteolytic cleavage of a precursor. Later, Honore et al.(35) cloned and expressed the cDNA encoding P-32, which extends beyond the 5` end of cDNA previously reported by Krainer et al., showing that ATG is the start codon at nucleotides 79-81, which is 12 nucleotides upstream of the 5` end of the sequence published by Krainer et al. However, the N terminus sequence of P-32 protein synthesized by cells infected with the Vaccinia virus construct of full-length cDNA including the conventional ATG start codon gave the N terminus amino acid sequence of P-32 as reported by Krainer et al., clearly defining the synthesis of P-32 as a proprotein of 282 amino acids, which is post-translationally processed by removal of the initial 73 amino acids to a mature protein of 209 amino acids. From our study, it is clear that the composite amino acid sequence of HA-binding protein containing 209 residues is completely identical to that of mature P-32 protein reported by both groups(24, 35) .
Celis et al.(37, 38) reported that the synthesis of P-32 protein is approximately 2-fold up-regulated in SV40-transformed human keratinocytes and MRC-5 fibroblasts, as compared with their normal counterparts, implying some transformation-dependent role of this protein. Our previous report has also shown a higher expression of HA-binding protein in AK-5, a histiocytic tumor cell line(22) . Recently, P-32 has been found to have 92% identity with a murine protein YL2 that modulates the effects of human immunodeficiency virus type 1 Rev(36) .
The open reading frame of the mature HA-binding protein predicted the protein as a highly acidic one with 28 glutamic acid (13.3%) and 20 aspartic acid (9.5%) residues having a molecular mass of 24 kDa. However, both the recombinant P-32 and HA-binding protein migrate with the same electrophoretic mobility of 34 kDa (Fig. 4A). This ambiguity may be explained due to its highly acidic nature, as an overestimation of molecular masses of highly acidic/charged proteins in SDS-PAGE is known(39, 40) . The predicted pI from the amino acid sequence is 4.04(24) , which has been also verified experimentally (Fig. 5B).
The mature HA-binding
protein showed several interesting features. The deduced amino acid
sequence of the protein demonstrated the presence of a motif, minimally
required for binding to HA, referred to as BXB,
where B is either R or K and X
is a stretch of
seven basic residues in between(10) . This protein also has one
such site
KLVRKVAGEK
, which contains one
extra glutamic acid residue. Peptide mimicry experiments (10) suggest that internal residues can vary by one amino acid
without having a detrimental effect on the ability of the protein to
bind HA. The presence of glutamic acid is also reported in the
HA-binding motif of TSG-6, a known HA-binding protein(41) .
Experimentally, the specific affinity of recombinant P-32 to HA was shown by its binding to biotinylated HA in a concentration dependent manner which can be competed only with excess unlabeled HA but not by RNA or DNA, ruling out its nonspecific anionic interactions. It may be mentioned here that though P-32 is co-purified with pre-mRNA splicing factor, Krainer et al. have already demonstrated that P-32 does not bind to RNA(24) . Furthermore, general RNA-binding motifs RNP1 and RNP2 are also absent in P-32 although present in the associated protein SF2. The observation that P-32 binds to HA in both reducing and nonreducing conditions suggests the lack of conserved cysteine residues in this protein in contrast to CD44 and cartilage link protein(42, 43, 44) . Rather P-32 behaves like RHAMM, which also lacks conserved cysteine residues and binds HA equally well under reducing conditions(45) .
Search
analysis further reveals the presence of a potential tyrosine sulfation
site (DCHY*PEDEV
) and three N-glycosylation sites (
WELELN*GTEA
,
VTFNIN*NSIPPTFD
, and
EWKDTN*YTLNT
) in the predicted amino acid
sequence. Incorporation of radiolabeled sulfate in CD44, a well known
HA receptor, is already reported(46, 47) . Moreover,
this protein has a proline-directed
PELTSTP
sequence, which may act as the substrate phosphorylation site of
protein kinases like extracellular signal-regulated kinase (ERK) (48) and cdc2 family (49) . It is already shown that
the Ser/Thr-Pro motif is sufficient for phosphorylation by ERK, and the
presence of an N-terminal proline residue at least 1 amino acid distant
from the phosphorylation site in the motif increases the efficiency of
substrate recognition(50) .
Sequence data further identifies
the mature protein as a multisite-phosphorylated protein, as it has one
protein kinase C phosphorylation site (DIFS*IREVS
) and five casein kinase II
phosphorylation sites (
LHT*DGDKAFVD
,
ESDIFS*IREV
,
EVSFQS*TGESEWKD
,
RGVDNT*FADELVEL
, and
LVELST*ALEHQEYI
). Several protein
substrates of casein kinase II have already been identified as
substrates of cdc2 kinase(51) , supporting our search data.
The presence of five casein kinase II phosphorylation sites in the
predicted sequence of HA-binding protein or P-32 protein may further
explain the association of P-32 to splicing factor SF2 in HeLa cells.
Casein kinase II is ubiquitously present in cytosol and the nucleus of
eukaryotic cells (52, 53, 54) and can also
behave as RNA-binding protein kinase(55) . The C group hnRNP
protein, implicated in splicing is known to get phosphorylated in
vivo by a casein kinase II activity (56, 57) .
Besides C group hnRNP, other pre-mRNA binding proteins, mainly UA2F,
the M 52,000 protein of trimeric
U
/U
/U
, and SF2, are
phosphoproteins(58) . Mayrand et al.(58) proposed that a cascade of critical phosphorylation and
dephosphorylation directs their sequential binding and release for
participation in the precatalytic state of splicing reaction. In the
context of complex association of SF2 and P-32 or HA-binding protein
and the presence of multiple casein kinase II phosphorylation sites in
P-32, we assume that phosphorylation in this protein may be regulating
the splicing ability of SF2. Krainer et al.(24) did
not rule out the possibility of a role of P-32 in splicing and rather
have shown the in vitro interaction between P-32 and splicing
factor SF2. (
)This observation has recently been supported
by the Peterlin group (36) confirming the interaction of YL2 or P-32
with the basic domain of HIV type I Rev, which is important not only
for its binding to Rev response element but also for its effect on RNA
splicing in vitro. In light of the new identity of P-32 as an
HA-binding protein, further investigations can be made to explain its
association with splicing factor SF2 as well as its role in modulation
of the function of human immunodeficiency virus type 1 Rev.