(Received for publication, October 23, 1995; and in revised form, February 23, 1996)
From the
Heparan sulfate proteoglycans and their corresponding binding
sites have been suggested to play an important role during the initial
attachment of murine blastocysts to uterine epithelium and human
trophoblastic cell lines to uterine epithelial cell lines. Previous
studies on RL95 cells, a human uterine epithelial cell line, had
characterized a single class of cell surface heparin/heparan sulfate
(HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were
isolated from cell surfaces by tryptic digestion, and partial
amino-terminal amino acid sequence for each peptide fragment was
obtained (Raboudi, N., Julian, J., Rohde, L. H., and Carson, D.
D.(1992) J. Biol. Chem. 267, 11930-11939). In the
current study, using approaches of reverse transcription-polymerase
chain reaction and cDNA library screening, we have cloned and expressed
a novel, cell surface HP/HS-binding protein, named HP/HS interacting
protein (HIP), from RL95 cells. The full-length cDNA of HIP encodes a
protein of 159 amino acids with a calculated molecular mass of 17,754
Da and pI of 11.75. Transfection of HIP full-length cDNA into NIH-3T3
cells demonstrated cell surface expression and a size similar to that
of HIP expressed by human cells. Predicted amino acid sequence
indicates that HIP lacks a membrane spanning region and has no
consensus sites for glycosylation. Northern blot analysis detected a
single transcript of 1.3 kilobases in both total RNA and
poly(A) RNA. Examination of human cell lines and
normal tissues using both Northern blot and Western blot analyses
revealed that HIP is expressed at different levels in a variety of
human cell lines and normal tissues but absent in some cell lines and
some cell types of normal tissues examined. HIP has relatively high
homology (
80% both at the levels of nucleotide and protein
sequence) to a rodent ribosomal protein L29. Thus, members of the L29
family may be displayed on cell surfaces where they may participate in
HP/HS binding events.
Heparin/heparan sulfate (HP/HS) ()proteoglycans
(HSPGs) expressed by different cells are able to interact with
HP/HS-binding effector proteins and perform important roles in
extracellular matrix structure and function, cell adhesion, growth, and
differentiation (Ruoslahti and Yamaguchi, 1991; Jackson et
al., 1991). HP/HS-binding effector proteins comprise a variety of
proteins that include growth factors (Ruoslahti and Yamaguchi, 1991),
extracellular matrix components (Kallunki and Tryggvason, 1992;
Vlodvasky et al., 1991), cytokines (Bernfield et al.,
1992), and cell adhesion molecules (Cole and Glaser, 1986).
Our laboratory has been interested in studying the mechanism of embryo implantation. We have found that HSPGs and their corresponding binding sites on cell surfaces may be important in the initial stage of mouse embryo attachment to uterine epithelium. Upon hatching from the zona pellucida, the embryo initially attaches to the uterus through the adhesion of the apical surfaces of the trophectodermal cells of the blastocyst. HSPGs are expressed by mouse embryos at the two-cell and post-implantation stages (Dziadek et al., 1985). Expression of HSPGs on trophectodermal cell surfaces of mouse blastocysts increases 4-5-fold at the peri-implantation stage (Carson et al., 1993; Farach et al., 1987), and HS expressed on mouse embryo surfaces is required for embryo attachment to isolated mouse uterine epithelial cells, fibronectin, and laminin (Farach et al., 1987). Similarly, studies have shown that the initial attachment of JAR cells, a human trophoblastic cell line, to RL95 cells, a human uterine epithelial cell line, is HP/HS-dependent, and enzymatic removal of HS from cell surfaces of both JAR and RL95 cells markedly inhibits JAR-RL95 cell-cell adhesion (Rohde and Carson, 1993). Therefore, HSPGs and their binding proteins also may play an important role in the initial attachment of human trophoblast cells to uterine epithelial cells.
Specific, saturable cell surface
HP/HS-binding sites have been identified on mouse uterine epithelial
cells and human uterine epithelial cell lines (Wilson et al.,
1990; Raboudi et al., 1992). Since mouse uterine epithelial
cells have only been available by primary cell culture, there is a
practical limitation to isolating HP/HS-binding sites from this source.
Effort has been placed on the study of HP/HS-binding proteins expressed
on the cell surfaces of a human uterine epithelial cell line, RL95
(Raboudi et al., 1992). Mild tryptic digestion of RL95 cell
surfaces removed most of cell surface HP/HS-binding activity. Three
major tryptic peptide fragments, ranging in M between 6,000 and 14,000, were released from cell surfaces and
retained HP/HS-binding activity. Partial amino-terminal amino acid
sequences from each of these three peptides were obtained (Raboudi et al., 1992).
We have employed an approach of reverse
transcription-polymerase chain reaction (RT-PCR) to identify
transcripts encoding cell surface HP/HS-binding peptides. Predicted
peptide sequence from one of the RT-PCR products revealed an antigenic
sequence that also has features of a HP/HS-binding motif suggested by
others (Cardin and Weintraub, 1989). Polyclonal antibodies directed
against the synthetic peptide corresponding to this motif recognize a
novel HP/HS-binding protein, named HP/HS interacting protein (HIP),
expressed on RL95 cell surfaces with an apparent M of 24,000 determined by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) (Rohde et al., 1996). This peptide selectively
binds HP/HS, recognizes certain forms of HP and cell surface HS
expressed by JAR and RL95 cells, and supports the attachment of human
trophoblast cell lines and a variety of other mammalian adherent cell
lines. (
)
Complete cDNA sequence of HIP has been isolated
by screening cDNA libraries using the partial cDNA sequence of RT-PCR
product of HIP. HIP cDNA sequence contains a single open reading frame
encoding 159 amino acids with a calculated molecular mass of 17,754 Da
and a predicted pI of 11.75. This protein is approximately 80%
homologous at both the nucleotide and amino acid level to a rodent
protein designated as ribosomal protein L29. Transfection of HIP into
NIH-3T3 cells results in expression of an M 24,000
protein that can be detected on the cell surface. Studies on the
expression and distribution of HIP revealed that HIP is expressed at
varying levels in a variety of human cell lines and normal human
tissues.
Total RNA from RL95 cells was isolated using the
method of RNA isolation of Xie and Rothblum(1991). RT-PCR was performed
on a DNA Thermal Cycler (Perkin-Elmer) using the protocol described in
the GeneAmp RNA PCR kit (Perkin-Elmer) with the concentration of
MgCl adjusted to 1 mM. The thermal cycle profile
described in the 3`-rapid amplification of cDNA ends protocol (Frohman,
1990) was followed. The cDNA pools from RT were amplified by PCR.
RT-PCR products were cloned into the pCR II vector using the TA Cloning
System kit (Invitrogen, San Diego, CA). Plasmids containing RT-PCR
products were isolated either following the method described by
Sambrook et al.(1989) or using ``Magic Minipreps''
DNA Purification System kit (Promega, Madison, WI). All of RT-PCR
products were sequenced using either the dideoxy-mediated chain
termination method (Sambrook et al., 1989) following the
procedure provided in the Sequenase version 2 kit (Amersham Corp.) or
automatic sequencing using fluorescently labeled sequencing primers (T7
and SP6) provided in the Applied Biosystems cycle sequencing kit and
analyzed on an Applied Biosystem model 373A automated sequencer
(Perkin-Elmer).
Figure 1: cDNA sequence and deduced amino acid sequence of HIP and comparison of HIP with mouse and rat L29. A, overlapping clones and RT-PCR product for HIP. The lines indicate the size and position of each clone and RT-PCR product. Restriction enzyme sites for Bsu36I (B), EcoRV (E), HindIII (H), and PstI (P) and positions of start codon, ATG, and stop codon, TAG, are indicated. B, cDNA sequence and deduced amino acid sequence of HIP. Deduced amino acid sequence is indicated below the nucleotide sequence. Nucleotides are numbered from the beginning of the cDNA sequence, and the deduced amino acid sequence is numbered from the beginning of the open reading frame. An in-frame stop codon is indicated with *, and the consensus polyadenylation signal, AATAAA, is underlined. HIP peptide sequence used for antibody production and HP/HS-binding activity study is shaded. Differences in nucleotide sequence among different clones are indicated with [ ]. C, comparison of amino acid sequence of HIP with mouse and rat L29. The deduced amino acid sequences of HIP and mouse and rat L29 are indicated. The HIP peptide sequence used for antibody production and HP/HS-binding studies is underlined. Asterisks denote the identical amino acid residues. Deletions or insertions are indicated by dashes.
Further analysis of the sequence showed that HIP cDNA contains a single open reading frame of 477 bp, starting with an ATG codon at position 28 with characteristic purines at positions -3 and +4 relative to the start ATG codon (Kozak, 1987), and ending with a stop codon TAG at position 506 (* in Fig. 1B). This open reading frame encodes a protein of 159 amino acid residues with a calculated molecular mass of 17,754 Da. The HIP peptide sequence used for antibody production is indicated in the predicted protein sequence (shaded letters in Fig. 1B). Following the open reading frame, there are 121 bp of a 3`-untranslated region that contains a polyadenylation signal (AATAAA) at nucleotide position 613. The predicted protein sequence has high content of positively charged amino acid residues (K + R = 29.6%) and a predicted pI of 11.75.
A comprehensive search of the GenBank, EMBL, and SWISS-PROT data bases revealed that the nucleotide sequence of HIP has 80.5% identity in 549-bp overlap to a rat mRNA for ribosomal protein related to yeast ribosomal protein YL43, 77.6% identity in 603-bp overlap to R. norvegicus (rat) mRNA for ribosomal protein L29, and 76.6% identity in 640-bp overlap to Mus musculus (murine) large ribosomal subunit protein mRNA. A BLAST homology search using GenBank revealed two human nucleotide sequences, designated as a putative human ribosomal protein L29, in GenBank (accession number U10248 and Z49148) showing the same nucleotide sequence as that of HIP cDNA; however, these sequences are not published and no further information is available. The predicted amino acid sequence of HIP has 80.3% identity in 157-amino acid residue overlap to a rat 60 S ribosomal protein, L29; however, the region encoding the peptide sequence (HIP peptide) used for antibody production and HP/HS-binding activity studies is not conserved among human and rat or mouse (Fig. 1C). Consequently, the antibodies are specific to human and do not cross-react with L29 of rat or mouse.
Figure 2:
Northern analyses of RNA from RL95 cells.
Approximately 3.5 µg of poly(A) RNA (lane
1) and 20 µg of total RNA (lane 2) were isolated from
RL95 cells and subjected to Northern blot analysis using
P-labeled cDNA of clone 23-1 (Fig. 1) as a
probe as described under ``Experimental Procedures.'' The
migration position of RNA standards (in kilobases) are indicated to the right, and the migration position of the 28 S rRNA is
indicated to the left.
Figure 3:
Expression of HIP on cell surfaces of HIP
cDNA-transfected NIH-3T3 cells. NIH-3T3 cells were transiently
transfected with HIP expression vector as described under
``Experimental Procedures.'' Transfected cells were grown on
coverslips for 24 h and fixed with 2.5% (w/v) paraformaldehyde and
immunostained with anti-HIP antibodies as described under
``Experimental Procedures.'' A and B are
photographs taken at low (96 ) and high (480
)
magnifications, respectively. In both A and B, arrows point to the HIP-transfected cells, and examples of
cells not transfected during transient transfection and lacking
staining are indicated with arrowheads.
Figure 4:
Western blot analysis of HIP expression in
HIP DNA-transfected NIH-3T3 cells. Total homogenates of RL95 cells (lane 1, 50 µg), parental NIH-3T3 cells (lane 2,
200 µg), and NIH-3T3 cells transiently transfected with HIP cDNA (lane 3, 200 µg) were separated on SDS-PAGE, and HIP
expression was detected using Western blot analysis as described under
``Experimental Procedures.'' Positions of the molecular mass
markers (in kilodaltons) as well as the migration of the M 24,000 HIP protein are
indicated.
Figure 5:
HIP mRNA expression and distribution among
human cell lines. A, total RNA (20 µg/each) from different
human cell lines were isolated, separated on 1% (w/v) agarose gel, and
subjected to Northern blot analysis using P-labeled cDNA
of clone 23-1 as a probe as described under ``Experimental
Procedures.'' The cellular sources of RNA is as follows: lane
1, AFb-11, normal fibroblast; lane 2, HeLa, cervical
epithelium; lane 3, HEC, uterine epithelium; lane 4,
HL60, leukemic; lane 5, HUVEC, normal umbilical vein
endothelium; lane 6, Ishikawa, uterine epithelium; lane
7, MDA-231, breast epithelium; lane 8, JAR, trophoblastic
epithelium; lane 9, NCI-H69, lung small cell; lane
10, RL95, uterine epithelium; lane 11, Tu138, lung
fibroblast; and lane 12, NIH-3T3, mouse embryonic fibroblast.
Positions of HIP mRNA and 28 S rRNA are indicated. B,
ribosomal RNA 28 S and 18 S were stained with ethidium bromide to show
equal loading of amount of RNA.
Figure 6: HIP expression and distribution in different human cell lines. Total proteins (100 µg/each) from different human cell lines were extracted and separated on SDS-PAGE, and HIP expression was determined by Western blot analysis: lane 1, HeLa, cervical epithelium; lane 2, 2774, ovary epithelium; lane 3, NCI-H69, lung small cell; lane 4, AN3-CA, lymph endometrium; lane 5, RKO, intestine epithelium; lane 6, Ter9113, trophoblastic; lane 7, Ter9117, trophoblastic; lane 8, Glioma2, glial; lane 9, Tu138, lung fibroblast; lane 10, NeuroB1, neural; lane 11, HL60, leukemic; and lane 12, JAR, trophoblastic epithelium. Positions of protein molecular mass markers are indicated.
In the present study, we have isolated and sequenced a
full-length cDNA encoding HIP, a novel human HP/HS-binding protein
expressed on cell surfaces. This cDNA encodes a protein of 159 amino
acids with high content of basic amino acids. There is no a potential
transmembrane domain present in the predicted amino acid sequence of
HIP. HIP is associated with the cell surface (Rohde et al.,
1996 and present article). Therefore, it is likely that HIP is a
peripheral membrane protein, perhaps bound to other proteins, lipids,
or polysaccharides. The predicted amino acid sequence of HIP does not
contain a classical hydrophobic amino-terminal signal peptide (Blobel
and Dobberstein, 1975); however, there are multiple reports of the lack
of a signal peptide in the sequences of membrane or secreted proteins
(Kikutani et al., 1986; Bettler et al., 1989; Brown et al., 1987). The predicted protein sequence of HIP predicts
a molecular mass of 17,754 Da. This is significantly less than expected
for M 24,000 protein recognized by
anti-HIP-peptide on SDS-PAGE. The anomalous molecular mass may be due
to the highly basic character of HIP (predicted pI = 11.75).
Other highly basic proteins, e.g. histones, migrate relatively
slowly on SDS-PAGE (von Holt et al., 1989; Weber and Osborn,
1975), apparently due to an inordinately high amount of SDS binding.
Alternatively, post-translational modifications may increase the size
of HIP. No consensus sites for glycosylation are evident in this
sequence, but other modifications are possible. Transfection of
full-length cDNA of HIP into NIH-3T3 cells resulted in the expression
of a protein with M
of 24,000 determined by
SDS-PAGE, further demonstrating that the cloned cDNA sequence encodes
the same protein recognized by the antibody and contains the predicted
peptide sequence. Northern blot and Western blot analyses revealed that
both the 1.3-kb mRNA and M
24,000 protein are
expressed coordinately in a variety of human cell lines. Differential
expression of HIP protein also has been observed in normal human
tissues examined (Rohde et al., 1996). (
)
Sequence comparison with available data bases revealed that HIP has a relative high similarity (80%) to rodent L29, a ribosomal protein, at both the nucleotide and protein sequence level. It is possible that HIP is the human homologue of rodent L29. It is noteworthy that in the mouse, L29 is a member of 15-18 genes or pseudogenes (Rudert et al., 1993). It is not clear what functions, if any, these sequences serve in rodents. Several lines of evidence indicate that HIP does not function simply as a ribosomal protein. First, HIP can be detected on cell surfaces of cells transfected with HIP cDNA or RL95 cells (Rohde et al., 1996). Second, HIP is expressed in a nonconstitutive fashion in different human cell lines and normal tissues. Constituent ribosomal proteins would be expected to be expressed at stoichiometric levels in different cells and with respect to the cellular content of rRNA species. While there is precedent for limited modulation of ribosomal proteins in some cases (Nomura et al., 1982; Rudert et al., 1993), these proteins are never essentially absent as in the case for both HIP mRNA and protein in cells like MDA-231 and NCI-H69. Collectively, these data strongly argue that HIP is not critical to ribosomal function.
Cell surface localization (Rohde et al., 1996) and
HP/HS-binding activity suggest that HIP may play a role in
HP/HS-involved cell-cell or cell-matrix interactions or have other
functions yet to be determined. In the studies of rodent L29, the
identification and localization of this protein was based on sequence
homology analysis and standard procedures of ribosomal protein
isolation (Ostvold et al., 1992; Svoboda et al.,
1992; Rudert et al., 1993). The distribution of the protein
was only examined in one study by Northern blot analysis and in
situ hybridization (Rudert et al., 1993). No studies of
the expression of the L29 protein are reported. Thus, it is of interest
to re-examine the expression of rodent L29 considering the possibility
that it may not be a ``housekeeping'' protein. Considering
the existence of the high number of sequences closely related to L29
(Rudert et al., 1993), it will be important to use probes
specific for each gene in such studies.
HIP may be expressed both at
cell surfaces and intracellularly. Several reports indicated that some
proteins are present both at cell surfaces or secreted as well as
inside the cell (Terada et al., 1995). These examples include
certain growth factors (Abraham et al., 1986; Jaye et
al., 1986), cytokines (Matsushima et al., 1986), and
lectins (Cooper and Barondes, 1990). Why these proteins are expressed
in both locales is unclear. In the present case, it is not known if
intracellular HIP is contained within vesicles or organelles or in the
cytoplasm. The following paper (Rohde et al., 1996)
demonstrates that almost all of the cell-associated HIP is formed in a
100,000 g sedimentable fraction and, therefore, is not
present in a freely soluble form. Several mechanisms for sorting of
cytoplasmic and secreted proteins have been postulated, including that
cell lysis, death, or leakage might be responsible for the release of
these proteins (D'Amore, 1990) or that the release might be
induced by plasma membrane evaginations (Cooper and Barondes, 1990).
Previous studies in our laboratory have suggested that HSPGs and their corresponding binding sites may play an important role in the initial attachment of mouse embryo to uterine epithelium. In the present study, a novel cell surface HP/HS-binding protein from a human uterine epithelial cell line has been cloned and expressed. The accompanying article (Rohde et al., 1996) describes expression of this protein in normal human lumenal epithelium, a location where HIP could participate in embryo attachment. Rigorous examination of a role for HIP in human embryo attachment is not possible; however, such studies can readily be performed in rodents. Therefore, it should be possible to identify the murine functional homologue of HIP and study the expression and physiological functions of this protein in the mouse in order to test its potential role in embryo implantation.