(Received for publication, June 30, 1995; and in revised form, August 22, 1995)
From the
The receptor for advanced glycation end products (RAGE), a
newly-identified member of the immunoglobulin superfamily, mediates
interactions of advanced glycation end product (AGE)-modified proteins
with endothelium and other cell types. Survey of normal tissues
demonstrated RAGE expression in situations in which accumulation of
AGEs would be unexpected, leading to the hypothesis that under
physiologic circumstances, RAGE might mediate interaction with ligands
distinct from AGEs. Sequential chromatography of bovine lung extract
identified polypeptides with M values of
12,000 (p12) and
23,000 (p23) which bound RAGE.
NH
-terminal and internal protein sequence data for p23
matched that reported previously for amphoterin. Amphoterin purified
from rat brain or recombinant rat amphoterin bound to purified sRAGE in
a saturable and dose-dependent manner, blocked by anti-RAGE IgG or a
soluble form of RAGE (sRAGE). Cultured embryonic rat neurons, which
express RAGE, displayed dose-dependent binding of
I-amphoterin which was prevented by blockade of RAGE
using antibody to the receptor or excess soluble receptor (sRAGE). A
functional correlate of RAGE-amphoterin interaction was inhibition by
anti-RAGE F(ab`)
and sRAGE of neurite formation by cortical
neurons specifically on amphoterin-coated substrates. Consistent with a
potential role for RAGE-amphoterin interaction in development,
amphoterin and RAGE mRNA/antigen were co-localized in developing rat
brain. These data indicate that RAGE has physiologically relevant
ligands distinct from AGEs which are likely, via their interaction with
the receptor, to participate in physiologic processes outside of the
context of diabetes and accumulation of AGEs.
Incubation of proteins or lipids with aldose sugars results in
nonenzymatic glycation and
oxidation(1, 2, 3, 4, 5, 6, 7) .
Following formation of the reversible early glycation products, Schiff
bases and Amadori products, further complex molecular rearrangements
result in irreversible advanced glycation end products (AGEs). ()Factors favoring nonenzymatic glycation include delayed
protein turnover, as in amyloidoses, accumulation of macromolecules
with high lysine content, and situations with elevated glucose levels,
as in diabetes. AGE formation occurs during normal aging, and at an
accelerated rate in diabetes, in which their accumulation in the plasma
and vessel wall has been speculated to underlie the pathogenesis of
vasculopathy (1, 2, 4) .
One of the
principal means through which AGEs impact on cellular elements is
through interaction with cellular binding proteins. Although there are
several possible cell-associated polypeptides with which AGEs might
interact(8, 9) , our work has focussed on the receptor
for AGEs (RAGE), as its expression in endothelium, vascular smooth
muscle, mononuclear phagocytes, and the central nervous system suggests
strategic loci for interaction with the glycated
ligands(10, 11) . The potential pathophysiologic
relevance of AGE-RAGE interaction was emphasized by studies
demonstrating that blockade of RAGE prevented multiple AGE-induced
perturbations of cellular functions. For example, AGE-stimulated
mononuclear phagocyte migration and activation, endothelial expression
of vascular cell adhesion molecule-1, and increased monolayer
permeability were prevented by blocking AGE interaction with
RAGE(12, 13) . Consistent with the latter data in cell
culture, in vivo studies have shown RAGE to mediate the early
and rapid removal of AGEs from the intravascular space, AGE induction
of vascular oxidant stress and vascular hyperpermeability in diabetic
animals and rodents infused with AGEs (14, 15, 16, 17) . ()These data emphasize a potential role for AGE-RAGE
interactions in pathologic states, and have led us to assess RAGE
expression in vasculopathies, especially that associated with diabetes.
During the course of tissue surveys to assess RAGE distribution, it became evident that expression of the receptor occurred in early development, especially in the central nervous system; i.e. in situations distinct from those in which AGEs might be present. RAGE is a member of the immunoglobulin superfamily of cell surface molecules (19) , and the extracellular domain consists of one putative ``V'' type followed by two putative ``C'' type domains, bearing closest homology to neural cell adhesion molecule(19) . In view of previous studies with other immunoglobulin-like receptors, such as intercellular adhesion molecule-1, in which one receptor has several pathophysiologically relevant ligands(20) , we considered the hypothesis that AGEs might be accidental ligands for a receptor which had other functions. By analogy with other immunoglobulin superfamily members, we speculated that RAGE might participate in cell-cell or cell-matrix interactions, or, perhaps, might function as a cytokine or growth factor receptor. Toward this end, we sought to define putative natural ligands for RAGE which were not AGEs. In this study we report the identification of amphoterin as a ligand for RAGE: amphoterin binds RAGE with higher affinity than AGEs; RAGE serves as the binding site for amphoterin on rat embryonic cortical neurons; amphoterin-RAGE interaction promotes neurite outgrowth in cell culture; and, RAGE and amphoterin are co-expressed in developing rat neuroepithelium. Although future in vivo studies will be required to determine the contribution of amphoterin-RAGE interaction in neuronal development, these studies provide a first step in identifying novel functions for RAGE distinct from its role as a receptor for AGEs in the vasculature.
Fractions
from the heparin column were screened for RAGE binding activity
following their adsorption to MaxiSorp microtiter wells (Nunc,
Naperville, IL) in bicarbonate/carbonate buffer (pH 9.6) for 16 h at 4
°C. After blocking excess binding sites in the wells with
phosphate-buffered saline containing bovine serum albumin (1%), a
binding assay with I-RAGE was performed. For these
studies, RAGE was purified to homogeneity from bovine lung, as
described(10) , and radioiodinated by the lactoperoxidase
method using Enzymobeads (Bio-Rad) according to the
manufacturer's instructions. The final specific radioactivity of
the tracer was 1,000 cpm/ng, and
I-RAGE was >95%
precipitable in trichloroacetic acid (20%), migrated as a single band
on SDS-PAGE (comigrating with the unlabeled protein), and bound AGEs.
I-RAGE was added to wells previously coated with
fractions from the heparin column for 3 h at 37 °C in
phosphate-buffered saline containing bovine serum albumin (0.2%). At
the end of the incubation period, wells were washed rapidly (10 s/wash
and 0.3 ml/wash) with Hank's balanced salt solution (Life
Technologies, Inc.). Bound radioactivity was eluted with NaCl (2 M) and counted in a
-counter (Pharmacia LKB,
Gaithersburg, MD). Fractions containing RAGE binding activity,
identified in the 0.5 M NaCl eluate, were dialyzed versus phosphate-buffered saline containing octyl-
-glucoside (0.1%;
final pH 7.4) and applied to Affi-Gel 10 (Bio-Rad), to which had been
bound purified bovine RAGE (2 mg/ml resin), according to the
manufacturer's instructions. After 16 h of incubation at 4
°C, the resin was washed with 10 bed volumes of buffer
(phosphate-buffered saline containing octyl-
-glucoside, 0.1%), and
eluted by high salt (NaCl, 2 M). Fractions containing binding
activity for
I-RAGE, based on the competitive binding
assay above, were dialyzed versus phosphate-buffered saline
containing octyl-
-glucoside (0.1%) and subjected to nonreduced
SDS-PAGE (10%). Protein on the gel was either visualized by silver
staining (Bio-Rad), or gels were sliced (2 mm each) and individual
slices were eluted by incubation in acetate buffer (pH 8.0) for 4 h at
4 °C. The mixture was then centrifuged to pellet debris, and
supernatants were tested for RAGE binding activity. Two positive
fractions were identified, eluted from the gel, and subjected to
nonreduced SDS-PAGE (12%). Protein on the gel was visualized by either
silver or Coomassie Blue staining to identify bands. Two protein bands
with M
23,000 and
12,000 were visualized
and sequence analysis performed as follows.
Figure 1:
Purification of RAGE binding proteins
by sequential chromatography on heparin-Sepharose (A) and RAGE
affinity chromatography (B), followed by preparative SDS-PAGE (C and D). A, bovine lung powder was
extracted with octyl--glucoside and chromatographed on
heparin-Sepharose. Specific binding of
I-RAGE alone
(total) or
I-RAGE in the presence of an 100-fold excess
of unlabeled RAGE (nonspecific binding) and OD
of the
fractions was determined. Specific
I-RAGE binding (total
minus nonspecific) is shown. B, fractions with RAGE binding
activity, corresponding to those eluted at an NaCl concentration of 0.5 M were subsequently chromatographed on Affi-Gel 10 to which
had been bound purified RAGE. After extensive washing with
equilibration buffer, the resin was eluted with NaCl (2 M) and
positive fractions were identified based on their ability to bind
I-RAGE. C, fractions with RAGE binding activity
were subjected to preparative SDS-PAGE (nonreduced, 12%) followed by
silver staining. D, lanes of the SDS gel identical to that in panel C were cut into 1-mm pieces, gel elution was performed
as described, and RAGE binding activity was
determined.
Purified 23-kDa polypeptide (p23) was subjected
to NH
-terminal and internal sequence analysis. The
NH
-terminal sequence of p23 had 17 identifiable residues of
the first 20. The 17 identified residues matched exactly the NH
terminus of bovine amphoterin (Table 1). Five internal
sequence peptides generated by Lys-C and cyanogen bromide cleavage were
found (Table 1). In total, the residues identified from the
NH
-terminal and five internal peptides (including tentative
assignments) matched 65 of 68 residues in amphoterin. All of the
inconsistent amino acid assignments occurred in one fragment (number
3), possibly the result of a contaminating peptide that copurified with
fragment 3 on the HPLC). Each of the internal peptides generally
aligned with amphoterin in a location that would be predicted by the
cleavage used to generate the peptide (Lys-C peptides follow lysine
residues and cyanogen bromide peptides follow methionine). These data
indicated that p23 and amphoterin were identical and suggested the
hypothesis that RAGE might be a cell surface acceptor site for
amphoterin.
Figure 2:
Binding of amphoterin to immobilized RAGE:
dose dependence (A), effect of sRAGE (B), blocking
antibody to RAGE (C), and AGE albumin (D). A, dose-dependence. Rat brain amphoterin was prepared and
purified as described and radiolabeled with I. Microtiter
wells were coated with purified RAGE (2.5 µg/well) and a binding
assay was performed as described by adding
I-amphoterin
alone (total binding) or in the presence of 100-fold excess unlabeled
amphoterin (nonspecific). Specific binding of
I-amphoterin is plotted versus free/added
I-amphoterin. Parameters of binding for rat brain
amphoterin were K
= 6.4 ±
1.0 nM with capacity of 46.7 ± 2.4 fmol/well. B, effect of sRAGE.
I-Amphoterin (3 nM) was
preincubated with the indicated concentration of sRAGE for 2 h, and
then binding to RAGE immobilized on microtiter wells was studied as
above. Maximal binding was specific binding observed in the absence of
added sRAGE. C, effect of anti-RAGE IgG. RAGE-coated
microtiter wells were preincubated with anti-RAGE IgG or nonimmune IgG
for 2 h and then a radioligand binding assay performed with
I-amphoterin and excess unlabeled amphoterin as above. D, effect of AGE albumin. RAGE-coated wells were preincubated
with AGE albumin or native albumin for 2 h and then a radioligand
binding assay with
I-amphoterin was performed as
above.
Figure 3: Immunostaining and in situ hybridization for RAGE protein and mRNA, respectively, in E17 rat cultured neuronal cells. Cortical neuronal cells were isolated from E17 rat embryos and cultured for 2 days on poly-L-lysine (50 µg/ml)-coated dishes. Cells were then fixed with paraformaldehyde (2%) for immunostaining or paraformaldehyde (4%) with Nonidet P-40 (0.1%) for in situ hybridization studies. Fixed cells were stained with anti-rat RAGE IgG (a) or nonimmune IgG (b). In situ hybridization was performed with digoxigenin-labeled RAGE riboprobes and detected with alkaline phosphatase-conjugated anti-digoxigenin antibody. Panel c, antisense probe, and panel d, sense probe. Scale bar, 50 µm.
Since
neonatal cortical neurons expressed RAGE, it was important to determine
if they bound amphoterin, and if this was mediated by interaction with
RAGE. Radioligand binding studies with I-amphoterin were
performed on cortical neurons isolated from neonatal rat brain (E17)
and cultured on poly-L-lysine-coated wells after brief
fixation in paraformaldehyde.
I-Rat brain amphoterin
bound specifically to cultured neurons in a dose-dependent and
saturable manner, with K
= 8.8 ± 2.4
nM and a capacity of 28.8 ± 2.8 fmol/well (Fig. 4A). The binding of
I-recombinant
amphoterin was very similar, with K
= 8.09
± 1.60 nM and capacity of 34.66 ± 2.34 fmol/well
(data not shown). Binding of
I-rat brain amphoterin to
cortical neurons was dependent on interaction with RAGE; addition of
sRAGE blocked binding in a dose-dependent manner (Fig. 4B) and pretreatment of cells with increasing
doses of anti-RAGE IgG also prevented binding, whereas nonimmune IgG
was without effect (Fig. 4C). Binding of
I-rat brain amphoterin to cortical neurons was also
inhibited in the presence of AGE albumin, whereas native albumin was
without effect (Fig. 4D). Similar results were observed
using recombinant radiolabeled amphoterin (data not shown).
Figure 4:
Binding of amphoterin to cultured rat
cortical neurons: dose dependence (A), effect of sRAGE (B), blocking antibody to RAGE (C), and AGE albumin (D). A, dose-dependence. Cortical neuronal cells were
isolated from E17 embryos as described and cultured for 2 days on
poly-L-lysine-coated wells (1 10
cells/well). After cells were fixed with paraformaldehyde (2%), a
radioligand binding assay was performed with
I-amphoterin
as described. The parameters of specific binding of
I-amphoterin were: K
= 8.8 ± 2.4 nM with capacity of 28.8
± 2.8 nM. B, effect of sRAGE.
I-amphoterin (3 nM) was preincubated with the
indicated concentration of sRAGE for 2 h, and then binding to cultured
neurons was studied as above. Maximal binding was specific binding
observed in the absence of added sRAGE. C, effect of anti-RAGE
IgG. Cultured cortical neuronal cells were preincubated with anti-RAGE
IgG or nonimmune IgG for 2 h and then a radioligand binding assay
performed with
I-amphoterin and excess unlabeled
amphoterin as above. D, effect of AGE albumin. Cultured
cortical neurons were preincubated with AGE albumin or native albumin
for 2 h and then a radioligand binding assay with
I-amphoterin was performed as
above.
Figure 5:
Neurite outgrowth assays and the effect of
RAGE blockade. Eight-chamber wells were coated with either amphoterin
(20 µg/ml, panels a-f) or poly-L-lysine (50
µg/ml, panels g-i) for 18 h. Cortical neuronal cells were
isolated from E17 rat embryos as described and fixed with
paraformaldehyde (4%) containing Nonidet P-40 (0.1%) and stained with
monoclonal anti-tubulin antibody. a-c, effect of sRAGE.
Amphoterin-coated wells and neuronal cells were pretreated with sRAGE
or bovine serum albumin for 1 h at 37 °C. a, neurite
outgrowth on amphoterin-coated wells alone; b, in the presence
of sRAGE (50 µg/ml); or c, in the presence of sRAGE (5
µg/ml). d-f, neurite outgrowth in the presence of
anti-RAGE F(ab`) or nonimmune F(ab`)
was
assessed: d, in the presence of nonimmune F(ab`)
(40 µg/ml) or anti-RAGE F(ab`)
(e, 40
µg/ml; or f, 4 µg/ml). g-i, neurite outgrowth
on poly-L-lysine coated wells and the effect of RAGE blockade. g, neurite outgrowth on poly-L-lysine alone or in the
presence of sRAGE (50 µg/ml, h) or anti-RAGE
F(ab)`
(40 µg/ml, i). Scale bar, 50
µm.
Figure 6: In situ hybridization studies co-localize RAGE and amphoterin mRNA in the developing rat nervous system. Sections from E17, P5, and P17 developing rat brain were harvested and prepared as described above. In situ hybridization was performed as indicated in the text in E17 cerebral cortex: a, RAGE antisense; b, amphoterin antisense; c, RAGE sense, and d, amphoterin sense, P5 cerebral cortex; e, RAGE antisense; and f, amphoterin antisense, P5 hippocampus: g, RAGE antisense, and h, amphoterin antisense and P17 cerebellum: i, RAGE antisense; and j, amphoterin antisense. In all other cases, sense controls for RAGE and amphoterin were negative (data not shown). Scale bar, 1 mm (panels a, b, c, d, g, h, i, and j); scale bar, 500 µm (panels e and f).
Immunohistochemistry studies further supported the co-localization of amphoterin and RAGE in the developing nervous system of the rat. RAGE and amphoterin protein were both present in the cerebral cortex of P5 rat (Fig. 7: a, staining with anti-RAGE IgG; (c, nonimmune rabbit IgG revealed no staining) and b, staining with anti-amphoterin IgG (d, nonimmune chicken IgG was negative)). Higher magnification views of the P5 cerebral cortex revealed that while the cell bodies of the developing neurons were intensely positive for RAGE and amphoterin, staining of the developing axonal processes was even more dramatic (Fig. 7, e and f, respectively, thick arrow). Similar results were observed in the hippocampus of P5 rats (Fig. 7, g and i, anti-RAGE IgG; and h and j, anti-amphoterin IgG).
Figure 7: Immunohistochemistry studies co-localize RAGE and amphoterin protein in the developing rat nervous system. Sections from P5 developing rat brain were harvested and prepared as described above. Immunohistochemistry studies were performed as indicated in the text in P5 cerebral cortex: a, anti-RAGE IgG; b, anti-amphoterin IgG; c, nonimmune rabbit IgG; and d, nonimmune chicken IgG. Higher magnification views of the P5 cerebral cortex are shown in e (anti-RAGE IgG) and f (anti-amphoterin IgG). Similar results were observed in the P5 hippocampus: anti-RAGE IgG (g and i) and with anti-amphoterin IgG (h and j). In all other cases, controls utilizing nonimmune IgG were negative (data not shown). Scale bar, 1 mm (panels a, b, c, d, g, and h); scale bar, 250 µm (panels e, f, i, and j).
Taken together, these data from in situ hybridization and immunohistochemistry studies suggest that cells likely to express RAGE and amphoterin in developing rat brain are in close proximity, potentially allowing RAGE-amphoterin interaction to mediate neurite outgrowth.
We initially anticipated that cellular binding proteins/receptors for AGEs would be analogous to the collagen-like heterotrimeric scavenger receptors for acetylated low density lipoprotein on mononuclear phagocytes which mediate cellular uptake of modified lipoproteins(29) . In fact, recent studies have shown that such scavenger receptors can interact with AGEs(9) . However, RAGE, the first AGE cellular binding protein to be characterized in detail, was most analogous to immunoglobulin-like receptors. Other functional properties of RAGE suggested it was not an effective scavenger: infused AGEs, removed from the circulation by endothelial RAGE, were, in large part, transported by a transcytotic mechanism to the subendothelium where they became associated with matrix and smooth muscle cell elements(16) . Consistent with these data, following infusion of AGE albumin, induction of oxidant stress, assessed by appearance of malondialdehyde epitopes in the vasculature, was present in endothelium, subendothelium, and smooth muscle cells(14) . Furthermore, since the interaction of AGEs with RAGE enhanced monocyte chemotaxis and activation, and increased expression of endothelial vascular cell adhesion molecule 1, it was plausible that under physiologic conditions, natural or non-AGE ligands might interact with RAGE to mediate, for example, adhesive functions critical for normal development(12, 13, 30) .
As lung is a rich source of RAGE, we considered this tissue a
logical place in which to identify natural ligands of RAGE. The data
presented in this study demonstrate that two polypeptides, with M values of
12,000 and
23,000 on SDS-PAGE,
bind AGEs. Whereas the
12-kDa polypeptide has an unique
NH
-terminal sequence and its initial characterization is
still under way, the
23-kDa polypeptide proved to be identical to
amphoterin based on extensive protein sequence data. Although the more
rapid migration of p23 purified from bovine lung is somewhat different
from the M
reported for amphoterin purified from
rat brain (
30,000; (23) ), in view of sequence identity and
recognition of p23 by anti-peptide antisera made to either amino acids
2-21 or 94-101 (data not shown), it is most likely that p23
arises from cleavage of amphoterin during the tissue extraction or
purification procedure. Consistent with this view, Northern analysis of
rat lung RNA using amphoterin cDNA showed a single band, which is
similar to that reported for amphoterin mRNA in rat brain (data not
shown).
More detailed studies of amphoterin-RAGE interaction,
performed with larger amounts of amphoterin purified from rat brain or
produced recombinantly with baculovirus, showed that binding of
amphoterin to RAGE was specific, saturable, and of higher affinity than
that previously reported for AGEs (K of
6
nM for amphoterin versus K
of
50
nM for AGE albumin). Domains in amphoterin mediating
interaction with RAGE appear to be unrelated to AGE-like epitopes, as
enzyme-linked immunosorbent assay of amphoterin preparations showed no
detectable AGE antigen and anti-AGE IgG had no effect on amphoterin
binding to RAGE (data not shown), although this antibody has been
previously shown to block interaction of AGE albumin and AGEs
immunoisolated from diabetic plasma with RAGE(14) .
Amphoterin was first identified by Rauvala and
Pihlaskari(23) , as an 30-kDa polypeptide selectively and
highly expressed in the developing rat central nervous system. Cultured
embryonic neurons plated on amphoterin-coated matrices formed neuritic
processes, suggestive of their differentiation, a critical step in the
overall design and maturation of the nervous system. RAGE expression in
cell bodies and axonal processes was first observed in the bovine
nervous system in motor neurons and in certain populations of cortical
neurons (11) , and has been extended in this study to include
the developing rat central nervous system. Consistent with these data,
embryonic rat cortical neurons which have been show to produce
amphoterin also expressed RAGE. Neuronal RAGE was functional, as
indicated by its capacity to bind amphoterin and to mediate
amphoterin-induced neurite outgrowth.
Previous studies have identified potential binding sites for amphoterin distinct from RAGE. Salmivirta and colleagues (31) showed that amphoterin binds to syndecam, a cell surface proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan chains, in cultured mouse mammary epithelial cells. However, anti-syndecam antibodies did not detect specific staining in neural tissues. At the mRNA level, Northern blots of RNA from mouse forebrain hybridized with a syndecam cDNA showed a band of 4.5 kilobases; the significance of the latter was unclear, as it is considerably larger than that detected in epithelial cells (2.6 and 3.4 kilobases) and has not been further characterized in the literature to date. In other studies, Mohan et al.(32) presented evidence that sulfoglycolipids (immunoreactive with the monoclonal antibody HNK-1) may interact with amphoterin in the developing nervous system based on solid phase binding assays. The present studies identify RAGE as a cellular binding site for amphoterin, based on binding and functional data, and provide the strongest evidence, thus far, for a putative amphoterin receptor in the developing central nervous system.
Expression of amphoterin has also been demonstrated in transformed cells(33) . Parkkinen and colleagues (33) showed that C6 glioma cells, HL-60 promyelocytes, U937 promonocytes, HT1080 fibrosarcoma cells, and B16 melanoma cells produced amphoterin. These investigators also demonstrated that amphoterin strongly enhanced the rate of plasminogen activation and promoted the generation of surface-bound plasmin by both tissue-type and urokinase-type plasminogen activators, suggesting a role for amphoterin in invasive neoplastic lesions(18) . Future studies will be required to determine if RAGE modulates any of these properties of amphoterin in the biology of neoplasia.
In summary,
these data suggest possible roles for RAGE under circumstances in which
the presence of AGEs is unlikely. Specifically, in the rat embryonic
nervous system, RAGE is highly expressed, and co-localizes, at the
level of antigen and mRNA, with the presence of amphoterin. As blocking
access to RAGE, with either excess sRAGE or anti-RAGE
IgG/F(ab`), prevents amphoterin-induced neurite outgrowth
in cell culture, it is tempting to speculate that RAGE mediates the
potential role of amphoterin in neuronal development. These
observations provide a first step in characterizing a novel aspect of
the biology of RAGE, and emphasize the importance of future in vivo studies to address the physiologic significance of amphoterin-RAGE
interaction, with respect to the development and maturation of the
central nervous system.