(Received for publication, July 10, 1995; and in revised form, September 5, 1995 )
From the
Podocytes are responsible in part for maintaining the size and charge filtration characteristics of the glomerular filter. The major sialoprotein of the podocyte foot process glycocalyx is a 140-kDa sialoprotein named podocalyxin. Monoclonal antibodies raised against isolated rabbit glomeruli that recognized a podocalyxin-like protein based upon size, Alcian blue staining, wheat germ agglutinin binding, and distribution in renal cortex were used to expression clone cDNAs from a rabbit glomerular library. On Northern blot the cDNAs hybridized to a 5.5-kilobase pair transcript predominantly present in glomerulus. The overlapping cDNAs spanned 5,313 base pairs that contained an open reading frame of 1,653 base pairs and were not homologous with a previously described sequence. The deduced 551-amino acid protein contained a putative 21-residue N-terminal signal peptide and a 26-amino acid transmembrane region. The mature protein has a calculated molecular mass of 55 kDa, an extracellular domain that contains putative sites for N- and O-linked glycosylation, and a potential glycosaminoglycan attachment site. The intracellular domain contains potential sites for phosphorylation. Processing of the full-length coding region in COS-7 cells resulted in a 140-kDa band, suggesting that the 55-kDa core protein undergoes extensive post-translational modification. The relationship between the cloned molecule and the monoclonal antibodies used for cloning was confirmed by making a fusion protein that inhibited binding of the monoclonal antibodies to renal cortical tissue sections and then raising polyclonal antibodies against the PCLP1 fusion protein that also recognized glomerular podocytes and endothelial cells in tissue sections in a similar distribution to the monoclonal antibodies. We conclude that we have cloned and sequenced a novel transmembrane core glycoprotein from rabbit glomerulus, which has many of the characteristics of podocalyxin. We have named this protein podocalyxin-like protein 1.
Podocalyxin is the major sialoprotein of the glycocalyx lining the foot processes of glomerular epithelial cells (podocytes) where it is thought to maintain foot process structure and function in part by virtue of its negative charge(1) . Podocalyxin was first identified by Kerjaschki, Sharkey, and Farquhar (1) as an Alcian blue staining 140-kDa sialoprotein. Subsequent studies have shown that the negative charge is contributed by sulfate as well as by sialic acid (2) and that podocalyxin is present on the surface of endothelial cells as well as glomerular epithelial cells(3, 4) .
The interdigitating foot processes of neighboring podocytes create the huge intercellular surface area for glomerular filtration. The importance of charge for maintenance of this structure has been demonstrated by experiments where charge neutralization with polycations or desialylation with neuraminidase is associated with loss of the interdigitating foot process structure of the podocyte(5, 6, 7) . Similarly, the induction of podocyte foot process effacement in rats by injection of puromycin aminonucleoside is accompanied by leakiness of the glomerular filter, podocyte foot process detachment, and a reduction in sialylation of podocalyxin(8, 9) .
From these
studies we have the concept of podocalyxin as an intensely negatively
charged molecule present in large amounts on the podocyte foot
processes and lining the surface of endothelial cells. At these sites
podocalyxin may function in part by charge repulsion to maintain the
distance between foot processes of neighboring cells and between
circulating cells and the endothelium (``anti-adhesion
molecule''). We report here the molecular structure of a
transmembrane glycoprotein that we have cloned and sequenced as part of
an effort to understand in molecular terms how the glomerular filter
works and how it becomes dysfunctional in children and adults with the
nephrotic syndrome. We have named this protein podocalyxin-like protein
1 (PCLP1). ()
For transfection
experiments COS-7 cells were plated at 2.7 10
cells/60-mm dish overnight in DMEM (BioWhittaker, Walkersville,
MD) with 10% newborn calf serum. Cells were washed once with serum-free
DMEM before DMEM with Lipofectamine (Life Technologies, Inc.) 6
µl/ml and 2 µg of either the PCLP1 mammalian expression
construct or pcDNA3 vector. After 6 h of incubation, one volume of DMEM
with 10% fetal calf serum and 10% newborn calf serum was added. Cells
were lysed and extracted at 24 h after transfection in 200 µl of
PBS containing 1% Triton X-100, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 5 mMN-ethylmaleimide, 2 mM EDTA, and 8 M urea. Protein content of the extracts were analyzed by a modified
Bradford technique (Bio-Rad, Richmond, CA).
The wheat
germ agglutinin purified fraction of PCLP1 (8 µl containing 16
µg of total protein) was mixed with 1 µl of 2% SDS and 1 µl
of 1 M 2-mercaptoethanol and kept in boiling water bath for 5
min. The mixture was diluted to 200 µl in 20 mM Tris-HCl,
pH 7.5. 20-µl aliquots of the above preparation were digested with
heparinases I and II, heparitinase mix (0.5 units each), chondroitinase
ABC (0.5 units), N-glycosidase F (0.4 units), O-glycosidase (2 milliunits), and neuraminidase (0.1 unit) by
incubating at room temperature overnight followed by an additional 3 h
at 37 °C (endo--galactosidase and N- and O-glycosidase were obtained from Boehringer Mannheim; the
other enzymes were obtained from Sigma). Multiple enzyme digestions
were done similarly by adding the enzymes simultaneously to the aliquot
of denatured PCLP1 buffer mix. For endo-
-galactosidase digestions,
8 µl of wheat germ agglutinin fraction was boiled as described
above but diluted with sterile distilled water.
Endo-
-galactosidase digestion was performed in 50 mM sodium acetate buffer, pH 5.8, with 5 milliunits of the enzyme.
Aliquots without the addition of any enzyme incubated under identical
conditions served as controls. After incubation an equal volume of 2
SDS gel loading buffer with 2-mercaptoethanol was added, and
the reaction mixtures were kept in boiling water bath for 5 min,
separated on SDS-PAGE, and immunoblotted with 4B3 and BB5 mAbs as
described above.
Figure 1:
a, immunostaining pattern of rabbit
renal cortex stained with mAb 5F7 (A) or 4B3 (B)
shows strong staining of glomeruli and weaker staining of the
endothelial lining of artery (a), arteriole (a*),
vein (v), and peritubular capillary (pc). Control
staining with mAb (BB5) showed no staining (not shown). b,
SDS-PAGE and Western blots of glomerular extract developed with mAb
5F7. Gels were run under reducing and nonreducing conditions and either
stained or blotted as follows. Lanes A, Coomassie blue stain
showing a negative staining band at 140 kDa; lanes B, Alcian
blue stain showing a major band at 140 kDa and also a lighter band at
about 125 kDa. Under reducing conditions, a major band is seen at about
140 kDa, and two bands are present below this major band at about 125
kDa; lanes C, blot showing that I-WGA recognizes
the 140-kDa band under nonreducing conditions and bands at 140 and 125
kDa under reducing conditions; lanes D, blot showing that
prior treatment of the blot with neuraminidase abolishes
I-WGA binding; lanes E, blot showing that
monoclonal antibody 5F7 binds a band at 140 kDa under nonreducing
conditions and two bands at about 140 and 125 kDa under reducing
conditions; lanes F, blot developed using a control monoclonal
antibody BB5 shows no band; lanes G, blot developed using a
positive control for a mAb against an unrelated protein GLEPP1 showing
a band at approximately 200 kDa. c, immunogold-labeled
electron photomicrographs of rabbit kidney developed using the mAb 4B3 (upper panel) or a control antibody (BB5) of the same IgG1
isotype (lower panel). The upper panel shows gold
labeling predominantly on the foot processes of the glomerular
epithelial cell (large arrowheads) and on the body of the
glomerular epithelial cell (arrow), as well as occasional
labeling on the glomerular endothelial cell (small
arrowheads). No specific labeling was seen using the control mAb.
The bar represents 1
µM.
Figure 2: Diagrammatic illustration of PCLP1 cDNA and derived protein structure. Top, diagrammatic representation of the cDNAs used to derive the PCLP1 nucleotide structure. Clone Jo3 is one of the cDNAs cloned with the mAbs 5F7 and 4B3. Clone RACE 10 was cloned by PCR techniques. The remainder of the cDNAs shown were obtained using labeled cDNAs as probes. All clones were sequenced in both directions. Clone GN2 contains a 60-nucleotide amino acid putative alternative splice region in the coding region (see below). Bottom, diagrammatic representation of PCLP1 protein structure derived from the nucleotide sequence and aligned with a Kyle-Doolittle amino acid hydropathy plot. A single putative 26-amino acid transmembrane region is shown (solid black box). The N-terminal domain contains a hydrophobic 21-residue putative signal peptide (horizontal striped box). In addition are shown a serine-proline-rich region (diagonal striped box), a putative alternate splice region (vertical striped box), three potential sites for N-linked glycosylation (arrows), a potential glycosaminoglycan attachment site (V), cysteines for possible disulfide linkage (C), acidic areas (lightly shaded boxes), and a highly acidic C-terminal region (darkly shaded box). The binding region for the mAbs is between amino acids 63 and 246 and is based on the distribution of the cDNAs identified with the mAbs. This region is known to be extracellular because the mAbs 4B3 and 5F7 bind to nonpermeabilized glomeruli.
Northern blot analysis was performed to determine the approximate length of the transcript and to show the pattern of mRNA tissue expression. Northern blots performed using a 3.5-kb PCLP1 clone showed a major band at approximately 5.5 kb with minor bands at 7.1 and 4.4 kb (Fig. 3). Screening of different tissues shows the relatively much greater amount of mRNA in glomerulus as compared with other tissues (Fig. 3). This relative distribution of PCLP1 mRNA is similar to PCLP1 protein distribution as assessed by immunofluorescence using the 5F7 and 4B3 mAbs.
Figure 3: Northern blot developed using a 3.5-kb PCLP1 cDNA as a probe. Glomerular RNA was loaded in lanes A (30 µg) and B (5 µg). Lanes C-H were each loaded with 30 µg of RNA from renal cortex (lane C), liver (lane D), lung (lane E), intestine (lane F), spleen (lane G), and muscle (lane H). The major transcript is at 5.5 kb, with minor bands at 4.4 and 7.1 kb. The intensity of the signal is comparable between lane B (5 µg of glomerular RNA) and lane C (30 µg of renal cortical RNA), confirming that the amount of transcript detected is highest in the glomerular RNA sample. The positions of the 28 S and 18 S ribosomal RNA bands are shown (arrowheads). The blot was stained with methylene blue prior to transfer to confirm comparable loading of RNA onto lanes A and C-H with 6-fold less RNA loaded onto lane B.
The full length of the nucleotides sequenced from the overlapping cDNA clones spans 5313 base pairs ( Fig. 2and Fig. 4). An initiator methionine (base pairs 304-306) was identified by the following criteria, (a) The amino acid sequence was consistent with Kozak's consensus sequence (first methionine in the open reading frame, purine in position -3)(25) ; (b) this methionine was followed by a 21-amino acid putative signal peptide containing 12 consecutive hydrophobic amino acids (ALALAALLLLLL) (Fig. 4)(26) ; (c) the presence of numerous CpG-rich ``islands'' is compatible with this region being 5`-untranslated sequence (underlined in Fig. 4)(27) .
Figure 4: PCLP1 nucleotide and derived amino acid sequences obtained from cDNA sequencing. The initiation methionine was identified as the first ATG in the open reading frame and obeys Kozak's consensus. The underlined nucleotides in the putative 5`-untranslated region represent probable CpG regions. The underlined N-terminal 21 hydrophobic amino acids represent a putative signal peptide. The double underlined 26 hydrophobic amino acids represent a putative transmembrane region. Potential sites of N-linked glycosylation (black triangles), O-linked glycosylation (dotted underlines), glycosaminoglycan attachment (dashed underlines), and disulfide linkage (black circles) are shown. The 60-nucleic acid sequence number 1324-1383 (lowercase) was found only in clone GN2.
A stop codon was found at base pairs 1957-1959, indicating the end of the open reading frame (Fig. 4). This would correspond to an open reading frame of 1653 base pairs or a total of 551 amino acids. If the putative signal peptide (21 amino acids) is cleaved off in post-translational processing, then the mature protein will be 530 amino acids long, and the N-terminal amino acid would be glutamine. This conclusion has not been confirmed by N-terminal sequencing. The putative PCLP1 protein has a calculated molecular mass of 54.9 kDa and a calculated isoelectric point of 4.8. A Blast search of the available data bases showed no significant similarities to published nucleotide or protein sequences.
Analysis of the derived amino acid sequence showed a single 26-amino acid hydrophobic sequence compatible with a single transmembrane region ( Fig. 2and Fig. 4). Immediately C-terminal to the putative transmembrane region are positively charged amino acids (HQRLSHRK) as is typically found at the cytoplasmic side of a transmembrane region(28) . This orientation relative to the cell membrane is also supported by the fact that the clone Jo3 isolated using the mAbs codes for the region of the molecule N-terminal to the transmembrane region and that these two mAbs also bind to nonpermeabilized isolated rabbit glomeruli as assessed by immunofluorescence (data not shown). Thus the region of the molecule N-terminal to the putative transmembrane domain must be extracellular.
There are several other features of the extracellular domain that appear to be noteworthy. A 24-amino acid span (amino acids 245-268) is very rich in serine and proline (21 of 24 amino acids) and lies in the middle of the extracellular domain ( Fig. 2and Fig. 4). There are 4 cysteines available in the extracellular domain for potential disulfide linkage to form two loop structures ( Fig. 2and Fig. 4). One clone (GN2) from the glomerular library contained an additional 60-nucleotide insert(1324-1384) coding for 20 additional amino acids ( Fig. 2and 4). This putative alternately spliced region contains an unusual series of 3 alternating glutamines followed by 3 alternating glutamic acids (QRQSQGEGETE). The extracellular domain has 5 doublets of acidic amino acids (281-282, 366-367, 430-431, 445-446, and 448-449) similar to clusters of acidic amino acids thought to mediate calcium binding in other proteins(33, 34) .
Figure 5: Demonstration that the PCLP1 sequence codes for a protein product that is recognized by mAbs used for cDNA cloning. A, indirect immunofluorescence of cryostat sections of rabbit cortex stained with mAb 4B3 after incubation with a PCLP1 fusion protein (left panel) and a control fusion protein (right panel). Both sections were photographed at the same exposure time. The PCLP1-GST fusion protein but not the GLEPP1-GST adsorbed out the binding of the mAb 4B3 to the kidney section. The bar represents 50 µM. B, Western blot of isolated rabbit glomerular extract analyzed by SDS-PAGE under reduced and nonreduced conditions. The blot was developed with the mAb 4B3 (lanes A), a control mAb BB5 of the same isotype as 4B3 (lanes B), the polyclonal antibody produced by immunizing a guinea pig with the purified PCLP1-GST fusion protein (lanes C), and the guinea pig preimmune serum (lanes D). Both the mAb 4B3 and the polyclonal guinea pig anti-PCLP1-GST fusion protein antibody recognize bands of the same size (140 kDa) under reducing and nonreducing conditions. C, indirect immunofluorescent photomicrographs of rabbit kidney developed with either the monoclonal antibody 4B3 (left) or the anti-PCLP1-GST fusion protein polyclonal antibody (right). Both antibodies show major staining in the glomerulus and also staining of peritubular capillaries. The bar represents 100 µM.
The PCLP1-GST fusion protein was also used to immunize guinea pigs to raise polyclonal antibodies. On Western blot both the polyclonal antiserum and mAb 4B3 recognized a major band at approximately 140 kDa under both reducing and nonreducing conditions (Fig. 5B). The fusion protein was able to immunoadsorb the binding of both polyclonal and monoclonal antibodies to PCLP1 as assessed by immunofluorescence and Western blot (Fig. 5, A and B). Furthermore both the polyclonal anti-PCLP1 fusion protein serum and mAb 4B3 showed identical staining on immunofluorescence of the glomerulus and peritubular capillaries to that seen with the monoclonal antibodies (Fig. 5C). We conclude that the cDNAs cloned code for a molecule with the same size and distribution as that recognized by the monoclonal antibodies.
Figure 6:
Transfection experiments performed to
evaluate processing and the apparent molecular mass of the PCLP1
protein as assessed by SDS-PAGE. Left,
[S]methionine-labeled T7 coupled reticulocyte
lysate transcription and translation products analyzed by SDS-PAGE
under reducing conditions. Lane A shows PCLP1 cDNA (base pairs
1-2943 in pcDNA3 vector) expressed by the reticulocyte lysate
system alone. Lane B shows PCLP1 cDNA expressed by the
reticulocyte lysate system with added canine pancreatic microsomal
membranes. Lanes C and D show control reactions
performed with the vector alone. This experiment shows that the
unprocessed PCLP1 protein migrates with an apparent molecular mass of
70 kDa. The presence of microsomal membranes results in an increase in
the apparent molecular mass to approximately 80 kDa. Right,
expression of the PCLP1 protein in COS-7 cells. Western blotting with
mAb 4B3 of extracts of COS-7 cells transfected with PCLP1 cDNA (base
pairs 1-2943 in pcDNA3 vector) (lane G) shows the
presence of a 140-kDa band that runs slightly higher than the band seen
from a glomerular extract from isolated rabbit glomeruli (lane
H). COS-7 cells alone (lane E) or COS-7 cells transfected
with vector alone showed no band (lane F). Western blot of the
same extract as that run in lane G but developed with a
control monoclonal antibody (BB5) showed no band (lane I).
These data clearly show that COS-7 cells transfected with PCLP1 cDNA
produce a 140-kDa protein.
Figure 7:
Glycosidase experiments performed to
evaluate the potential glycosylation of PCLP1. Western blots of digests
of WGA affinity purified PCLP1 after incubation with various
glycosidases are shown. PCLP1 (1.6 µg of total protein in 20
µl) was digested with the following enzymes at room temperature
overnight followed by an additional 3 h at 37 °C. Gels were run
under reducing conditions. Lanes A, J, and K, no enzyme; lane B, heparinases I and II and
heparitinase mix (0.5 units each); lane C, chondroitinase ABC
(0.5 units); lane D, heparinases I and II, heparitinase, and
chondroitinase ABC (0.5 units each); lane E, heparinases I and
II, heparitinase mix (0.5 units each), chondroitinase ABC (0.5 units), N-glycosidase F (0.4 units), and O-glycosidase (2
milliunits); lane F, N-glycosidase F (0.4 units); lane G, O-glycosidase (2 milliunits); lane
H, N-glycosidase F (0.4 units) and O-glycosidase
(2 milliunits); lane I, neuraminidase (0.1 unit); lane
L, Endo--galactosidase (5 milliunits). As positive controls
done in the same incubation mixture, Western blots showed that the
GLEPP1 protein (which is copurified with PCLP1 from isolated glomeruli
by WGA affinity chromatography) showed digestion with N-glycodidase, O-glycosidase, neuraminidase, and
chondroitinase ABC, thereby confirming that there were not inhibitors
for these enzymes present in the incubation mixture and that these
enzymes were active under the conditions used (data not
shown).
That PCLP1 binds Alcian blue and has potential
glycosaminoglycan attachment site(s) (Fig. 1B and Fig. 4) suggests it may be a proteoglycan. However, digestion
with heparinases (Fig. 7, lane B), chondroitinase ABC (Fig. 7, lane C), heparinase and chondroitinase ABC (Fig. 7, lane D), and endo--galactosidase (Fig. 7, lane L) yielded no significant change in
apparent molecular mass. Similar results were obtained for digestions
performed in the absence of SDS or 2-mercaptoethanol (data not shown).
As a positive control blots of the same digests were probed with mAb
4C3, which we have recently reported and used to clone the podocyte
membrane protein tyrosine phosphatase GLEPP1(12) . These
experiments clearly showed changes in molecular mass of GLEPP1 on
incubation with chondroitinase ABC, N-glycosidase F, O-glycosidase, and neuraminidase (data not shown). Thus the
conditions used allowed digestion in the same incubation mix of another
glomerular epithelial glycoprotein. This result suggests that the
glycosidic linkages of PCLP1 are not accessible to the glycosidic
enzymes, that PCLP1 might be an unusual glycoconjugate not susceptible
to the enzymes used, or that the post-translational increase in
apparent molecular mass is not due to glycosylation.
The identification of the nucleotide sequence for this major protein distributed on glomerular epithelial cell and vascular endothelial cell surfaces is an important step toward understanding the role this molecule may play in glomerular and endothelial cell biology and pathology.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35239[GenBank].
Note Added in Proof-During the review of this manuscript, the following short human sequence submissions to GenBank(TM) were found to be homologous to the 3`-untranslated region of PCLP1: T87928, R99975, R99976, H65205, T87719, and H64714.