(Received for publication, July 18, 1995; and in revised form, September 18, 1995)
From the
Peripheral myelin protein 22 (PMP22) is expressed in many tissues but mainly by Schwann cells as a component of compact myelin of the peripheral nervous system (PNS). Mutations affecting PMP22 are associated with hereditary motor and sensory neuropathies. Although these phenotypes are restricted to the PNS, PMP22 is thought to play a dual role in myelin formation and in cell proliferation.
We describe the cloning and characterization of epithelial membrane protein-1 (EMP-1), a putative four-transmembrane protein of 160 amino acids with 40% amino acid identity to PMP22. EMP-1 and PMP22 are co-expressed in most tissues but with differences in relative expression levels. EMP-1 is most prominently found in the gastrointestinal tract, skin, lung, and brain but not in liver. In the corpus gastricum, EMP-1 protein can be detected in epithelial cells of the gastric pit and isthmus of the gastric gland in a pattern consistent with plasma membrane association. EMP-1 and PMP22 mRNA levels are inversely regulated in the degenerating rat sciatic nerve after injury and by growth arrest in NIH 3T3 fibroblasts.
The discovery of EMP-1 as the second member of a novel gene family led to the identification of the lens-specific membrane protein 20 (MP20) as a third but distant relative. The proteins of this family are likely to serve similar functions possibly related to cell proliferation and differentiation in a variety of cell types.
The 22-kDa peripheral myelin protein (PMP22) ()is a
hydrophobic protein of 160 amino acids with four predicted
transmembrane domains(1, 2) . Point mutations in PMP22
and aberrant expression of the PMP22 gene are associated with
various hereditary peripheral motor and sensory
neuropathies(3) . In particular, the spontaneous mouse mutants Trembler and Trembler-J carry point mutations in the pmp22 gene(4, 5) . In humans, the majority of
patients suffering from the autosomal dominant demyelinating neuropathy
Charcot-Marie-Tooth disease type 1A (6) bear a 1.5-megabase
intrachromosomal duplication of chromosome 17p11.2-12 that
includes the PMP22 gene(7, 8, 9, 10) . Rare point
mutations in the PMP22 gene have also been found in
non-duplication Charcot-Marie-Tooth disease type 1A patients (11, 12, 13) and in the severe congenital
peripheral neuropathy Dejerine-Sottas syndrome(14) .
Furthermore, the relatively mild, recurrent peripheral neuropathy with
liability to pressure palsies is associated with the reciprocal
deletion to the Charcot-Marie-Tooth disease type 1A
duplication(15, 16) .
As anticipated from the phenotype of PMP22 mutant organisms, PMP22 is mainly expressed by myelinating Schwann cells in the peripheral nervous system (PNS) where it is incorporated into compact myelin(1, 17, 18, 19) . PMP22 mRNA and protein have also been found in motorneurons, and transcripts have been identified in various adult tissues, including the brain, intestine, lung, and heart(8, 20, 21, 22) . Furthermore, PMP22 mRNA expression is widespread during mouse embryonic development (23) . Tissue-specific expression and regulation of PMP22 is controlled by a complex genetic mechanism involving two alternative promoter sequences(24, 25) . While the distal promoter is specifically activated in myelinating Schwann cells, the more proximal promoter was found to be active in all known PMP22-expressing tissues(24) .
Tissue culture experiments using NIH 3T3 cells
and primary dermal fibroblasts revealed that PMP22 is up-regulated
under growth arrest conditions, e.g. serum deprivation or
density growth arrest, suggesting a potential role for PMP22 in cell
proliferation(20, 27) . ()In support of
this hypothesis, recent studies employing retrovirus-mediated gene
transfer of PMP22 into cultured Schwann cells suggest a pronounced
influence of PMP22 expression on the length of the G
phase (28) .
Based on these findings, it was proposed that PMP22 serves a general role in cell physiology and an additional specialized function in PNS myelin(29) . However, all known mutations affecting the PMP22 are associated with a phenotype restricted to the PNS, and no consistent abnormalities in non-neural tissues have been detected, even in genetically engineered mice that are completely devoid of PMP22(30) . The most likely explanation for these apparently contradictory results is to postulate specific mechanisms that can compensate for the lack of PMP22 in non-neural tissues. Such processes may involve molecules that are structurally and/or functionally related to PMP22. However, although the putative membrane topology of PMP22 is similar to the gap junction-forming connexin protein family (31) or the tight junction component occludin(32) , PMP22 does not display convincing amino acid sequence identity with any other known protein.
In this study, we report the identification of a PMP22-related transcript and the characterization of its encoded protein, which we have designated epithelial membrane protein-1 (EMP-1) based on its tissue expression pattern. EMP-1 and PMP22 are significantly related in their overall structure and primary amino acid sequences and define a novel gene family that also includes a more distant relative, the lens-specific membrane protein MP20(33) .
NIH 3T3 fibroblasts were cultured, and growth was arrested by serum deprivation as described previously(24) . Exponentially growing and growth-arrested cells were harvested into 5 M GT buffer.
Figure 1:
cDNA and
amino acid sequences of rat EMP-1. The 981 base pairs contain an open
reading frame of 480 base pairs starting with an ATG codon at
nucleotide 1 and terminating with a TAA stop codon at position 481. A
potential cleavable signal peptide is highlighted in boldface letters (residues 1-16). A single motif for putative N-linked glycosylation is present between the first and second
hydrophobic domains at asparagine 43 (underlined). The
sequence has been submitted to the EMBL GenBank under the
accession number Z54212.
Amino acids are numbered according to the cDNA-predicted polypeptide shown in Fig. 1. A C-terminal cysteine residue was added to the loop 1 peptide for coupling purposes. The peptides were coupled to keyhole limpet hemocyanin as described previously(17) . The conjugates were used to immunize New Zealand White rabbits with Freund's complete adjuvant, and the animals were boosted 4 times with 500 µg of peptide and incomplete adjuvant at 2-week intervals. Blood was taken from the animals, and serum was isolated. The activity of the immune serum was tested on the immunogen by solid phase ELISA.
Figure 2: Predicted EMP-1 structure and amino acid sequence comparison of PMP22/EMP-1 family members. A, amino acid sequence comparison between rat EMP-1 and rat, mouse, and human PMP22. Identical residues are boxed. B, hypothetical topology of EMP-1 in a lipid bilayer based on computer-assisted hydrophobicity plots and secondary structure predictions. Residues identical to rat PMP22 are filled. Most of the point mutations in PMP22 known to result in hereditary peripheral neuropathies (diamonds) are conserved at the corresponding positions in EMP-1. The Y-shaped symbol indicates a potential N-linked carbohydrate chain. C, amino acid comparison of the three known PMP22/EMP-1/MP20 family members. The residues conserved between MP20 and PMP22 or EMP-1 are shown in white on black.
Fig. 2B depicts a theoretical model of the EMP-1 protein structure based on the hydrophobicity profile and the suggested structure of PMP22. Filled circles represent identical amino acid residues that are shared by rat EMP-1 and rat PMP22, while divergent residues are shown as open circles. The positions of the amino acids in PMP22 known to cause hereditary motor and sensory neuropathies (21) when mutated are highlighted in the EMP-1 sequence as diamonds (Fig. 2B). Interestingly, all of these mutations lie within the putative membrane-spanning domains, and five of the six residues are conserved in rat EMP-1. The conservation of these amino acid residues suggests that they may be of functional significance.
Additional data base searches with the EMP-1 and PMP22 sequences revealed that both display 30% amino acid identity to the lens fiber cell protein MP20(33) . MP20 is a 173-amino acid protein with similar structural features to EMP-1 and PMP22 (Fig. 2C). If MP20 is compared with PMP22 and EMP-1 simultaneously, the amino acid identity increases to 36% including strongly conserved motifs in the putative transmembrane domains (Fig. 2C).
Figure 3:
In vitro transcription and
translation of EMP-1 and PMP22 cDNAs. The
[S]methionine metabolically labeled proteins
were separated by reducing 15% SDS-PAGE. pcDNA-1 was used as a control,
and no nonspecific proteins can be detected. The EMP-1 cDNA generated
an 18-kDa protein that shows a tendency to aggregate (EMP-1).
Transcription and translation of the EMP-1 cDNA in the presence of CMM
results in a reduced rate of migration of the protein (EMP-1 +
CMM). This reduced migration is reversed by deglycosylation with N-glycosidase F (EMP-1 deglycosylated). The PMP22 cDNA
generates an 18-kDa protein whose apparent molecular weight increases
by 4-6 kDa when the reaction is performed in the presence of CMM
(PMP22 + CMM). Treatment with N-glycosidase F reduced the
molecular mass back to 18 kDa (PMP22 deglycosylated). Neither EMP-1 nor
PMP22 are substrates for signal peptidase in vitro as
indicated by the identical migration rate of the unglycosylated (EMP-1,
PMP22) and deglycosylated translation products (EMP-1 deglycosylated,
PMP22 deglycosylated). Prolactin (a) is a substrate for signal
peptidase; approximately 50% of the protein is processed when
translated in the presence of CMM (c) (Promega technical
manual).
-factor was used as a control for N-linked
glycosylation and was completely modified in the reactions (b)
(Promega technical manual).
Figure 4: Tissue distribution of EMP-1 and PMP22 mRNAs in the rat. Northern blot analysis with a radiolabeled EMP-1 probe shows high expression of 2.8-kb transcripts in the cecum, colon, rectum, fundus, and ileum (a). Lower levels of expression are observed in the duodenum and jejunum of the small intestine and the corpus and pylorus of the stomach (a). Additional transcripts of 1.7 kb are found in the fundus, ileum, cecum, and colon (a). In extraintestinal tissues, EMP-1 mRNA levels are high in the skin, whereas in the brain and lung, expression is comparable with the duodenum (b; panels a and c are 20-h exposures, and panels b and d are 48-h exposures of the same blot). PMP22 mRNA is also highly expressed in the intestine (c); its 1.8-kb transcript is most prominent in the rectum and cecum, where expression is comparable with that of PMP22 in the lung (c, d). Ten µg of total RNA was loaded per lane, and equal loading was verified by ethidium bromide staining (data not shown).
All tissues expressing EMP-1 mRNA contain 2.8-kb EMP-1 transcripts. In some regions of the gastrointestinal tract, however, including the fundus, ileum, cecum, and colon, additional transcripts of approximately 1.7 kb hybridize with the EMP-1 cDNA (Fig. 4a). Prolonged washing of the blots at high stringency did not result in the preferential loss of one of the signals relative to the other (data not shown), hence, we favor the interpretation that both the 2.8- and 1.7-kb transcripts are derived from the EMP-1 gene. Further studies of the differently sized transcripts will determine if they result from the use of alternative polyadenylation sites or arise by alternative splicing.
The EMP-1-probed Northern blot was stripped and reprobed with labeled rat PMP22 cDNA (Fig. 4, c and d). The results show that the tissue distribution of PMP22 mRNA and EMP-1 mRNA is similar but that there are subtle differences in their relative expression levels. PMP22 and EMP-1 transcripts are co-expressed to high levels in the skin, fundus of the stomach, cecum, colon, rectum, and duodenum. However, while the EMP-1 mRNA level is relatively high in colon compared with the rectum, PMP22 mRNA is low. Furthermore, PMP22 mRNA is more prominently expressed in the lung than EMP-1 mRNA but is relatively underrepresented in the brain (Fig. 4). Neither EMP-1 or PMP22 transcripts could be detected by Northern analysis of liver RNA.
Figure 5: Regulation of EMP-1 and PMP22 mRNA expression by sciatic nerve injury and in cultured cells. A, Northern blot analysis of EMP-1 mRNA reveals an increased expression in the degenerating distal part of the injured sciatic nerve (4 days after nerve cut) compared with normal control nerve. PMP22 expression is considerably higher than that of EMP-1 in the normal nerve (1-h exposure using the PMP22 probe compared with 36 h for the EMP-1 probe). In contrast to EMP-1, PMP22 mRNA is dramatically reduced in the distal nerve after injury. B, cultured, mitogen-expanded primary rat Schwann cells (pSC) and D6P2T Schwann cells display reduced EMP-1 expression following forskolin treatment. In contrast, PMP22 mRNA expression is increased under the same conditions. C, serum starvation-induced growth arrest of NIH 3T3 cells results in reduced EMP-1 mRNA expression and an increase in PMP22 expression relative to exponentially growing cells. Northern blot analyses were performed on the same blot (10 µg of total RNA/sample), which was stripped between hybridizations.
Figure 6: Expression of EMP-1 protein in the rat intestine. A, two rabbit anti-EMP-1 peptide antisera raised against each of the putative extracellular loops 1 and 2 of EMP-1 recognize a 25-kDa protein in the corpus gastricum (50 µg of protein lysate analyzed by 12% SDS-PAGE and Western blotting). The immune reactivity of the anti-loop 1 antiserum was blocked by preincubation with 250 µg/ml of the immunogen but not by the loop 2 peptide. B, strong expression of EMP-1 protein is found in the stomach and large intestine, and lower levels are present in the lung. Detection of a signal in the small intestine requires prolonged reaction time of the enzymatic detection system (data not shown).
The most prominent expression of EMP-1 protein is seen in the stomach, with lower levels being detectable in the cecum and large intestine. Expression in the duodenum and jejunum of the small intestine is considerably lower than in the other regions of the intestinal tract, in accordance with the reduced mRNA levels found in these tissues ( Fig. 4and Fig. 6A). Very low levels of the 25-kDa EMP-1 protein can also be detected in the lung (Fig. 6A), spleen, and thymus (data not shown).
In addition to the 25-kDa protein, both EMP-1 antisera detect a similar array of larger proteins in the intestine (Fig. 6B and data not shown). The presence of these additional immunoreactive species varies from experiment to experiment and between tissues (data not shown). In general, the additional bands are most prominent in lysates containing higher amounts of EMP-1 protein. Since the two antisera are directed against independent regions of EMP-1 protein, these larger immunoreactive species are likely to represent aggregated molecules, a phenomenon frequently seen with highly hydrophobic proteins.
Although the level of EMP-1 protein observed in some tissues does not strictly correlate with EMP-1 mRNA expression, EMP-1 protein can only be found in tissues where EMP-1 mRNA expression is seen. No immunoreactive proteins are detected by either antiserum in lysates of the EMP-1 mRNA-negative liver (Fig. 6A).
Figure 7: Detection of transiently expressed EMP-1 in COS cells. COS cells were transiently transfected with an EMP-1 expression construct and subsequently analyzed by immunofluorescence using the anti-loop 2 antiserum and a fluorescein isothiocyanate-labeled goat anti-rabbit Ig. Identical results were obtained using the anti-loop 1 antiserum (data not shown). The scale bar represents 20 µm.
Figure 8: Immunofluorescent localization of EMP-1 protein expression in the corpus gastricum. a, schematic view of the gastric mucosa showing the proliferative zone in the neck/isthmus region of the gastric gland and the migration of the differentiating epithelial cells toward the gastric pit. b, low magnification view of the gastric mucosa labeled with the polyclonal anti-EMP-1 loop 2 antiserum and detected with a Texas Red-labeled donkey anti-rabbit antibody. Intense immunoreactivity can be detected in the epithelial cells of the outer mucosa. No immunoreactive cells can be found toward the base of the gastric pit or in the submucosal muscle layer (sm). The intense labeling of the isolated cells at the base of the gastric mucosa is not specific, as it is also present in control sections incubated with preimmune serum (not shown). c, higher magnification of the labeled epithelial cells in the pit region. The migrating, differentiating epithelial cells in the isthmus express high levels of EMP-1 protein. d, transmitted light view of the region shown in panel c. e, cross-section through the gastric pit shows intense plasma membrane-associated labeling of the epithelial cells but no labeling of the mesenchyme. No staining is seen in transverse sections across the base of the gastric gland (not shown). f, transmitted light view of the section shown in panel e. The scale bars shown are 100 µm for b and 60 µm for panels c-f.
Ten-µm frozen sections of corpus gastricum were stained with anti-loop 2 antiserum. Strong immunoreactivity was detected in the outer epithelial cells of the gastric mucosa from the tip of the vilus down toward the isthmus and neck of the gastric gland (Fig. 8, b and c). In transverse section, the EMP-1 immunoreactivity appears to be associated with the plasma membrane of epithelial cells in the gastric pits (Fig. 8e). The epithelial cells deeper in the gastric gland show little or no immunoreactivity, and specific labeling was not detectable in the base of the gastric gland or in the submucosal muscle layer (Fig. 8b).
We report the cloning and characterization of the epithelial membrane protein EMP-1, a hydrophobic polypeptide of 160 amino acid residues. Computer-aided analysis revealed that EMP-1 shows 40% amino acid identity to PMP22, a PNS myelin protein that is responsible for inherited peripheral neuropathies. EMP-1 and PMP22 display similar hydrophobicity profiles, suggesting that both proteins contain four membrane-associated, potentially membrane-spanning domains. Thus, we propose that EMP-1 and PMP22 are two members of a gene family that also includes the lens fiber cell protein MP20, one of the major protein components of the mammalian eye lens(33, 40, 41) , as a distantly related third family member.
The high degree of identity at the amino acid level suggests that EMP-1 and PMP22 may serve similar functions. Close examination of the amino acid sequences of these proteins reveals that the hydrophobic regions, in particular the first two transmembrane domains, are highly conserved, suggesting that they are of particular functional importance. This hypothesis is further supported by the finding that the hydrophobic domains are the most strongly conserved regions between PMP22 species homologues. Interestingly, the amino acid residues in PMP22 that are sites of mutation in hereditary peripheral neuropathies are located within putative transmembrane domains, and the majority of these mutated amino acid residues are also conserved at the corresponding positions of EMP-1 and MP20.
The N-terminal signal peptides of both EMP-1 and PMP22 contain consensus sequences for signal peptidase cleavage. However, the signal sequence of PMP22 is not cleaved efficiently in myelinating Schwann cells as demonstrated by N-terminal sequencing of purified PMP22 protein(37) . Since the N terminus of EMP-1 is also not cleaved when synthesized in the presence of CMM, we hypothesize that the EMP-1 signal peptide is not removed during biosynthesis in vivo. This situation is reminiscent of the structurally related connexin family of gap junction proteins, where a specific mechanism has been postulated to prevent aberrant N-terminal processing(31) . Furthermore, MP20 does not contain a signal peptide cleavage consensus sequence, and N-terminal sequencing has shown it to be unmodified at its N terminus in vivo(42) .
The most interesting conservation within the putative extracellular domains of EMP-1 and PMP22 concerns the consensus sequence for an N-linked glycosylation. This glycosylation site in PMP22 carries a modified carbohydrate chain containing the L2/HNK-1 epitope, a structure which has been implicated in cell-cell recognition and adhesion processes (for recent review see Schachner and Martini(1995)) (43) . Although the presence and nature of carbohydrate moieties linked to EMP-1 remains to be determined, an N-linked glycosylation in the identical position of EMP-1 may be involved in cell recognition processes in the epithelium of the intestine.
EMP-1 and PMP22 are co-expressed in a wide range of tissues, and particularly high levels of transcripts for both proteins are found in the intestinal tract. The gastrointestinal tract is characterized by a continual and rapid renewal of its epithelial surface that continues throughout the animal's life. Pluripotent stem cells anchored in the isthmus/neck regions of the gastric gland give rise to progeny displaying increased proliferation and reduced potentiality, which progress to terminally differentiated mature cells(44) . During this differentiation process, the cells are highly migratory, with proliferation, migration, and differentiation all being tightly coupled. EMP-1 is found mainly in the proliferation and differentiation zones of the outer gastric gland as well as in the mature epithelial cells of the gastric pit region. In these cells, EMP-1 appears to be associated with the plasma membrane, with no clear distinction between the basal, apical, and lateral aspects.
PMP22 has been suggested to play a role in the control of
cell proliferation. In support of this hypothesis, evidence has been
presented that modulation of PMP22 levels in cultured Schwann cells has
a pronounced influence on the cell cycle(28) . In these
experiments, overexpression of PMP22 increased the length of the
G phase, while reduced expression resulted in a decrease.
Whether there is a similar effect of EMP-1 expression on the cell cycle
of epithelial cells remains to be determined.
In this report, we
confirm previous results that PMP22 is up-regulated in NIH 3T3
fibroblasts by growth arrest. In contrast, we show that EMP-1 mRNA
levels are down-regulated under the same conditions. A short cDNA
fragment corresponding to part of the 3`-untranslated EMP-1 mRNA has
been isolated previously and, in agreement with our results, has been
shown to be up-regulated in 3T3 fibroblasts 8 h after serum-induced
entry into the G phase(45) . The same conditions
lead to a decrease of the PMP22 mRNA level(20) . In addition,
we also observed that EMP-1 mRNA is up-regulated in the proliferating
cells of the distal stump of the rat sciatic nerve 4 days after cut
injury while PMP22 is down-regulated. Thus, PMP22 expression is induced
by growth arrest in various experimental paradigms, while EMP-1
expression is decreased. This conspicuous inverse regulation of PMP22
and EMP-1 during the cell cycle lends further indirect support to a
role of this protein family in the control of cell quiescence and
proliferation. This hypothesis is particularly intriguing given the
clinical interest in colon biology due to the prevalence of carcinomas
in this region of the intestinal tract.
We have identified the major lens fiber protein MP20 as a distant member of the PMP22/EMP-1 gene family. MP20 has been described as a lens-specific membrane protein that co-localizes with connexin 46 in fiber cell junctions, suggesting a role in organizing the junctional plaques(46) . MP20 has been shown to be absent from proliferating epithelial cells of the lens, with expression becoming prominent in differentiating as well as in mature lens fibre cells(33, 46) . Although the function of MP20 has not been elucidated, a role in signal transduction has been postulated, since MP20 is phosphorylated by cAMP-dependent kinase in vitro and binds calmodulin in overlay assays(47, 48) .
With respect to the biological function of the PMP22/EMP-1/MP20 protein family, it is informative to discuss the genetics of PMP22. PMP22 has been intensively studied due to its association with the most common forms of hereditary peripheral neuropathies. Based on these studies and evidence provided by spontaneous and artificially generated PMP22-defective mice, it is established that PMP22 plays a crucial role in the development and maintenance of peripheral nerves. No obvious abnormalities outside of the nervous system are found in patients carrying heterozygous duplications, deletions, or point mutations affecting the PMP22 gene or in transgenic mice completely devoid of PMP22. The latter findings are difficult to reconcile with the widespread expression of PMP22 and its potential role in cell proliferation. However, the identification of the PMP22/EMP-1/MP20 gene family offers a plausible hypothesis for this apparent paradox. The function of PMP22 in non-neural tissues may be partially redundant, and compensatory mechanisms may be at work in mutant organisms. Correct PNS myelination appears to be the obvious exception to the rule, since the function of PMP22 in this tissue is critical. It is conceivable that the exquisite sensitivity of the PNS to PMP22 gene dosage, as inferred from human genetics and transgenic mice, does not allow for compensation. Alternatively, PMP22 is extremely highly expressed by myelinating Schwann cells, and the regulatory elements controlling potential compensatory genes may not be able to adjust expression accordingly in the mutant organisms. This hypothesis is also supported indirectly by the complex regulation of the PMP22 gene involving both a Schwann cell-specific and a ubiquitously active promoter(24) . However, it cannot be excluded that PNS-specific functional peculiarities of the PMP22 protein are responsible for its crucial role in peripheral nerves.
These findings are reminiscent of the crystallin protein family (reviewed in (26) ). Crystallins are expressed in many tissues as enzymes or stress proteins. Through various evolutionary processes, they have subsequently been recruited as structural components of the lens. In some cases, high expression in the lens has been achieved using tissue-specific promoters or enhancers. Whether such mechanisms also apply to other known or yet to be found PMP22/EMP-1/MP20 family members remains to be seen.
In conclusion, PMP22 has been widely regarded as mainly a structural component of PNS myelin. Our description of EMP-1 and the concomitant identification of the PMP22/EMP-1/MP20 gene family, the expression pattern of these proteins in mainly epithelial tissues, and the observed differences in regulation of PMP22 and EMP-1 during the cell cycle support the concept that these proteins play multiple roles in cell biology. These functions may be related to both the switch from proliferation to differentiation as well as the maintenance of critical functions in the differentiated state.