From the Laboratory of Biochemistry and Molecular Genetics,
Lindsley F. Kimball Research Institute, New York Blood Center, New
York, New York 10021 and the § Program in Developmental Biology, the
Hospital for Sick Children and Department of Molecular and Medical
Genetics, University of Toronto, Ontario M5G 1X8, Canada
Received for publication, August 17, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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Ammonium transporters play a key functional role
in nitrogen uptake and assimilation in microorganisms and plants;
however, little is known about their structural counterpart in mammals. Here, we report the molecular cloning and biochemical characterization of Rh type B glycoproteins, human RhBG and
mouse Rhbg, two new members of the Rh family with distinct tissue
specificities. The RhBG orthologues possess a conserved
12-transmembrane topology and most resemble bacterial and archaeal
ammonium transporters. Human RHBG resides at chromosome
1q21.3, which harbors candidate genes for medullary cystic kidney
disease, whereas mouse Rhbg is syntenic on chromosome 3. Northern blot and in situ hybridization revealed that
RHBG and Rhbg are predominantly expressed in
liver, kidney, and skin, the specialized organs involving ammonia
genesis, excretion, or secretion. Confocal microscopy showed
that RhBG is located in the plasma membrane and in some intracellular
granules. Western blots of membrane proteins from stable HEK293 cells
and from mouse kidney and liver confirmed this distribution.
N-Glycanase digestion showed that RhBG/Rhbg has a
carbohydrate moiety probably attached at the NHS motif on exoloop 1. Phylogenetic clustering, tissue-specific expression, and plasma
membrane location suggest that RhBG homologous proteins are the long
sought major ammonium transporters in mammalians.
Ammonia transporters
(Amt)1 constitute a
superfamily of structurally divergent transmembrane (TM) proteins found
in diverse organisms of the three domains of life, Bacteria, Archaea,
and Eucarya. These proteins play a key functional role in the uptake and assimilation of ammonium ion (NH4+)
as a source of nitrogen in vast nitrogen-fixing microorganisms and
plants (1). The best-known Amts are those that are only recently
characterized in bacteria, yeast, and the flowering plant Arabidopsis thaliana (2-10). Whole-genome sequencing also
has revealed the presence of Amt-like proteins in archaeons (11, 12)
and nematode Caenorhabditis elegans (13). In a single given
species of lower organisms and plants,
NH4+ transport is often endowed with
multiple separate gene and protein forms. Members or subgroups of the
Amt superfamily vary significantly in primary structure, in number of
TM segments, and in kinetics of NH4+
uptake (2-10), thus correlating the function with environmental adaptation. Targeted gene replacements have shown that the absence of
Amt results in growth defect of the mutant organism when the culture
medium is depleted or lowered in NH4+
(2, 3, 6).
Instead of being a key compound of nitrogen acquisition in
microorganisms or plants, NH4+ is formed
as an end product of nitrogen metabolism in ammonotelic animals
and serves as an important urinary buffer in mammals. Mammalian species
such as rats, dogs, and humans face a net acid load and excrete
NH4+, via the kidney, to remove excess
protons to regulate systemic acid-base balance (14). Hence, the
maintenance of NH4+ homeostasis bestows
a vital mechanism in regulating net acid excretion. In human kidneys,
for example, half of the ammonia produced is excreted under normal
conditions and three-fourths of that is excreted in response to even a
mild acidosis (15). Active NH4+
transport in mammals has been well documented physiologically, at least
in the case of kidneys (16), but its building block remains to be
identified. A recent data base search (17) has revealed a marginal
homology between some Amt and red blood cell (RBC) Rh proteins
(particularly RhAG) (18-20), raising the possibility that the Rh
proteins may be an Amt equivalent in animal erythrocytes.
Although the RBC Rh proteins may serve to trap ammonia in circulation
(21), they are not appreciably expressed in liver, kidney, and skin
(22), the three major organs specialized in ammonia genesis, excretion,
or secretion. Nevertheless, RhAG homologues are rooted deeply in
evolution and occur in primitive life forms: the slime mold
Dictyostelium discoideum (23), marine sponge Geodia
cydonium (24), nematode C. elegans (13), and
fruit fly Drosophila melanogaster (23). Cross-reactions with
monoclonal anti-Rh antibodies suggest the presence of erythroid Rh-like
constituents in tissues of human and other mammals (25). Given these
observations, we undertook to isolate novel Rh homologues from
mammalian nonerythroid tissues. This has led to recent identification
of RhCG and Rhcg as first members of the nonerythroid Rh subfamily and
as a candidate ammonium transporters expressed in kidney and testis
(26). Nonetheless, the observed tissue specificity of RhCG suggested
the existence of additional Rh homologues that are putatively involved
in ammonium transport.
In this report, we describe the molecular cloning and biochemical
characterization of Rh type B glycoproteins, RhBG
and Rhbg, two new members of the Rh superfamily from human and mouse
nonerythroid tissues. We provide biochemical as well as evolutionary
genetic evidence for RhBG and Rhbg to specify a highly conserved
orthologous gene group that is distinct from both erythroid RhAG and
nonerythroid RhCG homologues. Furthermore, we show that the RhBG/Rhbg
orthologous pair is unique in primary amino acid sequence, in
chromosomal location, and in tissue specificity at the level of
mRNA and protein expression. The structural relationship,
phylogenetic clustering, organ-specific distribution, and plasma
membrane localization suggest that the RhBG proteins may be the long
sought major ammonium transporters in human and other mammals.
Cloning of Mouse Rhbg and Human RhBG cDNAs--
A BLAST
search (27) with the mouse Rhag cDNA (22) as a query
identified a homologous expressed sequence tag (clone AA798527) from
the mouse skin tissue. Gene-specific primers (GSPs) in either sense (s)
or antisense (a) were designed to isolate the mouse Rhbg
gene by the method of rapid amplification of cDNA ends (RACE). For
5'-RACE, 1 µg of cellular total RNA prepared from mouse liver was
reverse-transcribed with GSP-E4a1 (5'-CCAGCTGGGATCTGTAGAGGA-3'). The
resultant partial cDNA product was tailed with dCTP, as described in the RACE kit (version 2.0, Life Technologies, Inc.), and then amplified twice with supplied adapter primers and GSP: E4a2
(5'-CGTGAGAGGAACAGCCCGAAGTAG-3') and E4a3
(5'-CCAAATGTGTGAATTGTCATGGAC-3'), respectively. The 3'-RACE reaction
employed two GSP: E3s1 (5'-ATCTCTTTCGGGGCTGTTCTG-3') and E4s1
(5'-TGAGAGATGCTGGAGGGTCC-3'). To clone human RhBG, two degenerate primers (5'-TC(T/C)ATGAC(C/T)AT(C/T)CACAC(C/A)TTTGG-3' and
5'-GTT(G/A)TG(G/A)AC(A/T)CC(G/A)CATGTGTC-3') were used in reverse
transcriptase-PCR of liver total RNA. The primers define the conserved
part of TM6 and endoloop 5 in Rhbg. The 477-bp human RhBG band of the
mouse Rhbg counterpart was cut from native 5% PAGE and sequenced. New
and exact GSP were then designed for RACE in both directions.
Full-length Rhbg and RhBG were assembled using their GSP located in the
5'-untranslated region (UTR) and 3'-exon 10 (E10), respectively.
Sequence Analysis and Structure Prediction--
The nucleotide
and amino acid sequences of RhBG or Rhbg were analyzed with the
ClustalW program, Kyte-Doolittle hydropathy plot, or Chou-Fasman
secondary structure algorithm packed in LaserGene software (DNASTAR). A
dendrogram was constructed by multiple sequence alignment using
ClustalW, and amino acid sequence identity/similarity was derived from
pairwise comparison using the J. Hein method in Megalign software.
Chromosomal Mapping by in Situ Hybridization and Linkage
Analysis--
The human RHBG gene was localized by
fluorescence in situ hybridization (FISH), as described
previously (28). The genomic probes used for FISH were two human BAC
DNA clones (421G19 and 506J9), each containing an intact
RhBG gene, as fingerprinted by exon-specific
PCR.2 The mouse
Rhbg gene was assigned using the AflII
restriction fragment length polymorphism along with the Jackson BSS
interspecific backcross panel ((C57BL/6jEi x SPRET/Ei) x SPRET/Ei)
(29). The AflII restriction enzyme made an extra cut in
Rhbg intron 8 of the SPRET/Ei strain but not the C57BL/6jEi
strain. Genomic DNA of the 94-progeny panel was amplified with GSP: E8s
(5'-AGCCTGCAGAGTGTGTTTCC-3') and E9a (5'-AAACCTGGTCCTCGAAGCATTG-3').
The distribution of the AflII polymorphism in the 94 progeny
was computed to establish the linkage of the Rhbg gene with
known genetic markers.
Northern Blot Analysis of RhBG and Rhbg Gene
Expression--
Human and mouse Northern blots
(CLONTECH) retaining poly(A)+ RNA
prepared from various tissues were hybridized with the
32P-labeled RHBG and Rhbg cDNA
probes, respectively. The human RHBG probe was 519-bp long
and covered codon 1-173, whereas the mouse Rhbg probe was
849-bp long and spanned codon 173-455. The blots were hybridized and
washed under highly stringent conditions. The human RNA in Situ Hybridization to Mouse Embryos and Adult
Tissues--
RNA in situ hybridization was carried out as
described previously (30). A 405-bp cDNA spanning the 3' portion of
the mouse Rhbg gene (nucleotides 964-1368, see GenBankTM
accession number AF193808) was cloned in pCRScript SK(+) vector
(Stratagene). To prepare 33P-labeled RNA hybridization
probes, the above recombinant plasmid was made linear by either
BamHI (antisense direction) or NotI (sense
direction) digestion and then transcribed in vitro by T7 and
T3 RNA polymerases, respectively.
Construction of Expression Vectors--
Full-length RhBG was
cloned in pCR2.1 vector using Pfu DNA polymerase and GSP:
E10a(XhoI)
(5'-CCGCTCGAGTTAGGCCTGAGTGTCTGCCTC-3') and
E1s(BglII)
(5'-GAAGATCTGAGATCGCAGCCCAACCCATG-3'). All expression constructs were based on this plasmid and sequenced to preclude spurious mutations. To tag the green fluorescence protein (GFP) gene,
RhBG was amplified with E1s(BglII) and E10a(XhoI)
or E1s(BglII) and E10a(SalI), and inserted in the
pEGFP-C1 or -N3 vector (CLONTECH). To generate RhBG
C-tail (amino acids 416-458) expression constructs, the corresponding
coding region (nucleotides 1248-1377) was amplified by
E9s(BamHI) (5'-CGGGATCCAAGCTACCCTTTCTGGACT-3')
and E10a(XhoI). The cDNA fragment was purified and
cloned separately in pGEX-4T1 (Amersham Pharmacia Biotech) and
pET30a(+) (Novagen). For translation and expression studies,
full-length RhBG was also cloned separately in the pYES2 and
pcDNA3.1/MycHisA vector (Invitrogen) using compatible BamHI and XhoI sites.
Production of Polyclonal Antibodies against Human RhBG and Mouse
Rhbg Proteins--
The RhBG C-tail was expressed as a
glutathione S-transferase or a His6-fusion
peptide in Escherichia coli BL21 cells and purified as
described previously (26). To raise human RhBG antisera, five
injections of glutathione S-transferase-RhBG (300 µg/each) in rabbits followed the standard method (31). To raise mouse Rhbg
antisera, two short peptides, acetyl-AKGQRSATSQAVYQLFC-amide (amino
acids 377-392, corresponding to part of exoloop 6) and acetyl-CTETQRPLRGGESDTRA-OH (amino acids 440-455, specifying the extreme C terminus), were synthesized. They were purified, linked to
keyhole limpet hemacyanin, and used for immunization in rabbits (31).
All the antisera were affinity-purified before use.
Cell Culture, cDNA Transfection, and Confocal
Microscopy--
HEK293, HepG2, and HeLa cells (ATCC) were grown in
Dulbecco's modified Eagle's medium, as described (26). Trypsinized
cells were seeded on a 6-well plate or coverglass (MatTek), cultured for 24 h, and then transfected with LipofectAMINE (Life
Technologies). For confocal imaging, 3 × 105 cells
plated on coverglass were transfected with 1 µg of
RHBG-GFP or GFP-RHBG plasmid and cultured for
24 h. GFP was excited at 488 nm with argon laser, and the light
emitted between 506 and 538 nm was recorded for the fluorescein
isothiocyanate filter. Images were collected with a Bio-Rad MRC600
confocal scan head on a Nikon Eclipse 200 microscope and were processed
with Adobe Photoshop (version 4.0).
In Vitro Transcription-coupled Translation of Human
RhBG--
Full-length RhBG plasmids were used as DNA templates for
in vitro transcription-coupled translation with a Promega
kit and [35S]methionine (15 mCi/ml, Amersham Pharmacia
Biotech). RhBG cloned in pYES2 carried no tag, whereas that cloned in
pcDNA3.1/MycHisA vector had a 3' in-frame fusion with the
c-myc epitope sequence and six His codons.
35S-Labeled RhBG or RhBG-Myc, treated with or without
canine pancreatic microsomal membranes (CPMM)(Promega), was analyzed by
12% SDS-PAGE.
Stable Expression of Human RhBG Protein--
To attain stable
expression, ~3 × 105 HEK293 cells were transfected
with the RHBG-myc construct. Stable clones were selected in
Dulbecco's modified Eagle's medium containing G418 (800 µg/ml) and
isolated as described previously (32).
Membrane Protein Preparation, N-Glycanase Digestion, and Western
Blotting Analysis--
Membrane proteins from HEK293 stable cells,
human RBC, or mouse liver, kidney, and heart tissues were prepared as
described (33, 34). Protein was resuspended at 1 mg/ml in ice-cold
buffer (10 mM HEPES, pH 7.5, 1 mM
MgCl2, 250 mM sucrose), and an aliquot was
taken for N-glycanase digestion (PNGase F, New England
BioLabs) as specified by the supplier. The glycanase-digested and
native proteins were subjected to 12% SDS-PAGE and blot-transferred
onto a Hybond membrane. Western blots were incubated with various
primary antibodies specific for RhBG or Rhbg, which are denoted in
figure legends. Protein bands were visualized using a chemiluminescent detection kit (Pierce).
Primary cDNA and Amino Acid Structures of RHBG and
Rhbg--
The longest open reading frames were 1377 bp for human
RHBG and 1368 bp for mouse Rhbg, which encode a
polypeptide of 458 and 455 amino acids, respectively (Fig.
1). In both cDNAs, the 5'-UTR lacks a
Kozak consensus (35), whereas the 3'-UTR contains an atypical
polyadenylation signal, GATAAA (see GenBankTM accession numbers
AF193807 and AF193808). RHBG or Rhbg has a very high G/C content (60% versus 58%), notably different from
RHAG or Rhag (43% versus 42%) (20,
22), but similar to RHCG or Rhcg (58%
versus 55%) (26). At the protein level, RhBG and Rhbg are
85% identical and 94% similar, the highest among all Rh protein pairs
known to date. RhBG differs from Rhbg mainly by a 9-bp insertion (GCCGCGGGC for 8AAG10 at the N terminus), a
longer 3'-UTR, and 26 nonconserved substitutions scattered on the
polypeptide backbone (Fig. 1). Whole-protein composition analysis is
also highly comparable between RhBG and Rhbg, and both proteins possess
a molecular mass of ~49.5 kDa and a net negative charge at
physiological pH (pI 6.70 versus 6.64). RhBG and Rhbg each
have a single NX(S/T) N-glycosylation motif
(49NHS51 versus
46NHS48, Fig. 1) and thus are possibly
expressed as glycoproteins. The noted structural features and high
sequence identity define Rhbg and RhBG as a conserved orthologous group
and suggest that the two proteins perform the same function in mice and
humans.
Comparison of RhBG and Rhbg with Erythroid and Nonerythroid Rh
Members--
Sequence comparison showed that RhBG/Rhbg is homologous
to both erythroid RhAG/Rhag and nonerythroid RhCG/Rhcg pairs (Fig. 1).
This is largely due to a conservation of hydrophobic regions that may
define the TM domains and adjacent residues. Notably, four blocks of
sequences, the extreme N and C termini, the portion around the
NX(S/T) motif, and the predicted exoloop 6, are very divergent among the three pairs. Like RhCG (26), RhBG is much less
similar to the Rh polypeptides (18, 19).
Secondary structure analysis predicted RhBG to be a polytopic protein
with 12 putative TM domains (Fig. 2).
This topology is a conserved fold, because no gap occurs in the
sequence spanning TM2-11 of all six proteins (Fig. 1). Notably, the D
and E negative charges predicted to reside in TM4 or -5 are conserved,
whereas the E to Q change in TM1 or -5 alternates among the three Rh
protein pairs (Fig. 1). RhBG may be similar to RhAG with N and C
termini facing the cytoplasm (36), but its TM profile and secondary structure are more akin to RhCG than RhAG (Fig. 2). The secondary structural homology arises mainly from an increased exoloop size and
higher sequence identity between RhBG and RhCG, although the two
proteins differ entirely in their extreme C-terminal sequences (Fig.
1). Together, the results indicate that RhBG and Rhbg are new members
of the Rh protein family possibly having functional properties distinct
from other mammalian Rh homologues.
Relationships between the Rh Family and the Amt Superfamily--
A
connection of some Amt to erythroid Rh proteins was noted previously
(17). However, due to the limited number of known sequences, the
evolutionary and structural relationships between the Rh and Amt
families were not clear. A similarity search using RhBG and Rhbg as
queries showed that they were directly related to certain bacterial and
archaeal Amt proteins (see below). A thorough analysis of available Rh
(this study and Ref. 26) and Amt protein sequences resulted in a better
definition of their relationship.
As shown in Fig. 3, members of the Amt
superfamily are distributed in three major clusters. Cluster I consists
of two subclasses, one from bacteria and archaeons (nos. 1-24) and the
other from fungi, including yeast (nos. 27-32). Two Amt from A. thaliana (nos. 25 and 26) are also grouped here and may, in
analogy to archaeal Amt (nos. 8-11), result from horizontal genetic
transfers (11, 12). Cluster II (nos. 48-59) is, intriguingly, composed of Amt members from diverse organisms of the three domains of life,
including invertebrates. This cluster, with its branch point predating
both the Rh family and Cluster I, might have served as a provider of
ancestors for the evolution of the latter two clusters. Cluster III
(nos. 62-70) is comprised of highly homologous Amt members that are
present in plants only and occur as high affinity
NH4+ transporters. Notably, the vast
majority of Amt proteins are polytopic membrane proteins with 10-12
TM-spanning segments.
With regard to members of the Rh family (nos. 33-47), they form a
single distinct cluster that intercepts Clusters I and II of the Amt
superfamily (Fig. 3). This clustering placed all Rh members as a late
divergent group possibly originating from one or more primitive Amt
genes that had pre-existed in Cluster II. All but the slime mold (a
protozoan) homologue, Dd.RhgA, are of metazoan origins, including the
most primitive living metazoan (sponge), invertebrates, fish, and
mammals. These observations reinforce a structural as well as a likely
functional relationship between the Rh and Amt proteins. In the case of
human RhBG, it most resembles the Amt members from Cluster II that are
present in cyanobacteria and archaeons, respectively (Fig. 3). It is
noted that 1) despite an extensive search, no Rh homologue other than Amt is found in bacteria, archaeons, fungi, or plants; and 2) Rh and
Amt coexist in the slime mold, nematode, and possibly fruit fly (13, 23, 26 and this study).
Structural Homology and Divergence between RhBG and Amt
Proteins--
Detailed sequence analysis further revealed the features
and structural homology between the RhBG and Amt proteins. One such example is shown in Fig. 4. RhBG bears a
similar degree of overall homology to AmtA and Amt1 from two different
cyanobacteria species. A comparable extent of sequence identity was
noted between some divergent members within the Amt superfamily itself
(3). Although the three proteins only share 57 identical amino acid
residues, RhBG is characterized by a composite nature in many other
sites, having sequence identities with either AmtA or Amt1 (Fig. 4). This feature leads to a much higher overall sequence similarity and is
also evident when RhBG is aligned with other Amt from archaeons or
bacteria (see Fig. 3, nos. 8, 12, and 56 for examples). Moreover, many
substitutions are conservative in nature or similar to the consensus of
Rh glycoprotein homologues, including some 20 G to A or A to G changes
(Fig. 4). Of further significance is that the secondary structure or
12-TM topology is conserved between RhBG and the two Amt, particularly
with regard to their internal portions.
Nevertheless, as revealed by sequence alignment (Fig. 4), a number of
structural differences between RhBG and the two Amt are worth
mentioning. 1) Several major gaps are evident, although they are likely
involved in variable surface loops. 2) The E/D negative charges
conserved in the TM domains of various Rh glycoprotein homologues from
mammals (Fig. 1 and data not shown) are not seen in the two Amt
proteins. 3) The amino acid identity is dispersed largely in a patched
manner, although the sequence similarity runs in longer stretches. 4)
The Rh family members possess unique signatures (26) that are absent
from the Amt family. Taken together, the structure homology and
sequence divergence may embody a conserved mechanism for
NH4+ movement and a differential
coupling in glutamine (and/or urea) synthesis, respectively.
Chromosomal Assignment of Human RHBG and Mouse Rhbg Genes--
To
define the location of the human RHBG gene, the genomic DNA
isolated from BAC clones was labeled and used as FISH probes to paint
interphase chromosomes. The FISH result showed that RHBG resides at 1q21.3 of human chromosome 1 (Fig.
5A). This recognized a
trans relationship of RHBG with RHCG
at 15q25 (26) but a cis unlinked relationship with
RHCED, the locus at 1p34-36 encoding Rh blood groups (37).
Notably, RHBG lies within the candidate region for autosomal
dominant medullary cystic kidney disease (OMIM174000) (38). By linkage
analysis, Rhbg showed no recombination with the
Bglap1 marker (logarithmic odd score, 28.3). This
placed Rhbg distal to Mab21/2 but proximal to
Npr1 on mouse chromosome 3, where many markers are syntenic
to human 1q21 containing RHBG (Fig. 5B).
Northern Blot Analysis of RHBG/Rhbg Expression--
Northern blot
analysis confirmed RHBG or Rhbg expression only
in nonerythroid tissues (Fig. 6). In
human adult, RHBG was expressed as one major form in kidney
but multiple forms in liver and ovary (at a moderate level) (Fig.
6A). These mRNA species arose from alternative splicing
events involving two distinct Alu repeats present in intron
1 of the RhBG gene.2 In human fetus, RHBG was
expressed relatively strongly in kidney but only weakly in liver (Fig.
6A). With regard to mouse Rhbg, it was comparably
expressed in kidney and liver (Fig. 6B, left). However, unlike the human counterpart, mouse Rhbg expression
produced a single mRNA form and was not subjected to alternative
splicing, suggesting a differential regulation. Although the ovary and
skin tissues were not examined, they had identical expressed sequence tags of Rhbg as detected by BLAST search (AI406901,
AI011329, and AA798527). Rhbg transcripts were also evident
in mouse embryos at 15- and 17-day gestation (Fig. 6B,
right). Thus, in a temporal order, Rhbg
expression is later than erythroid Rhag or Rhced
(22) but earlier than nonerythroid Rhcg (26).
RNA in Situ Hybridization--
RNA in situ
hybridization provided further data on the sites of Rhbg
expression in mouse embryos and adult tissues. Consistent with the
pattern of gestational expression (Fig. 6B), Rhbg
showed a strong signal in the kidney (not shown) and skin but a
moderate signal in liver of 16.5-day embryos (Fig.
7A). In adult skin, Rhbg was highly expressed in dermal hair follicles and
papillae (Fig. 7, B and C). In adult kidney,
Rhbg was widely and abundantly distributed in the cortex and
the medulla (Fig. 7D). Higher magnification suggested that
Rhbg is mainly present in the epithelial linings of the
renal convoluted tubules and Henle's loops (Fig. 7, E and F). In adult liver, the Rhbg signal was dispersed
in a dotted fashion (Fig. 7G) and was evidently confined to
hepatocytes (Fig. 7, H and I). Combined with its
plasma membrane location (Fig. 8),
RhBG/Rhbg may mediate NH4+ passage for
glutamine (or urea) synthesis inside hepatocytes. Together, the data
show that the pattern of Rhbg expression does not overlap
with that of Rhcg (26) in complex nonerythroid tissues.
Localization of RhBG Protein at Subcellular Level--
The
subcellular location of RhBG was defined by confocal imaging of
transiently expressed RhBG-GFP fusion proteins (Fig. 8). Control cells
(panels A, D, and G) showed an even
distribution of green fluorescence in the cytoplasm. However, the cells
transfected with RhBG-GFP (panels B, E, and
H) or GFP-RhBG (panels C, F, and I) displayed green fluorescence that was condensed mainly in
the plasma membrane and in some intracellular granules. Time-lapse recording revealed a dynamic movement of those granules (not shown), likely indicating transport of RhBG from intracellular vesicles to the
plasma membrane. Notably, the same imaging pattern was observed in both
homologous cells (panels B, C, E, and
F) and heterologous cells (panels H and
I). Thus, similar to RhCG (26), the membrane biogenesis of
RhBG is not cell-type specific. These results suggest that, contrary to
the RBC Rh polypeptides (39), nonerythroid Rh proteins possess
intrinsic topogenic signals necessary for their transport and insertion
into the plasma membrane.
RhBG in Vitro Translation and Processing in Microsomal
Membranes--
Because RhBG has only one
49NHS51 sequon (Fig. 1), predicted to
reside in the exoloop 1 (Fig. 2), its glycosylation status was assessed
by in vitro translation with CPMM incubation. In the absence
of CPMM, in vitro translated RhBG, whether or not carrying the C-terminal Myc epitope and His6 tags, migrated as a
single band (Fig. 9A). By
SDS-PAGE analysis, the untagged and tagged RhBG species were estimated
to have an apparent molecular mass of 38-40 and 42-44 kDa,
respectively. The untagged RhBG was smaller than the predicted RhBG in
size; this anomaly probably resulted from the high hydrophobicity of
the protein. Nevertheless, with added CPMM, the in vitro
translated RhBG, in both untagged and tagged forms, increased in size
and migrated as a broader band (Fig. 9B). The difference
between CPMM-treated and untreated RhBG appeared to match the size of a
single N-linked glycan. These results indicate that the RhBG
polypeptide underwent appropriate targeting, translocation, and
processing (e.g. N-glycosylation) in microsomal
membrane compartments.
RhBG/Rhbg Protein Expression in Human Stable Cell Lines and in
Mouse Native Tissues--
To establish if RhBG is expressed as a
glycoprotein in vivo, membrane proteins were isolated from
stable HEK293 cells harboring the transfected RHBG-myc gene.
Digestion with PNGase F followed by Western blot analysis confirmed
RhBG to be an N-glycosylated membrane protein. As shown in
Fig. 10A, the two blots
probed with anti-RhBG C-tail antisera (left panel) and
anti-Myc monoclonal antibody (right panel), respectively,
displayed a seemingly identical banding pattern. Moreover, the
deglycosylated RhBG-Myc from HEK293 cells (Fig. 10A,
lanes 5 and 6) appeared in the same size as
in vitro translated RhBG-Myc (Fig. 9A, lane
6), suggesting that the same translation initiator functions
in vivo and in vitro. Nevertheless, the size of
the N-glycosylated product from stable expression was larger
than that of the glycosylated species induced by CPMM incubation (Fig.
9B). This observation implies that RhBG is more efficiently
glycosylated under in vivo conditions.
To analyze the expression and biochemical properties of Rh type B
glycoprotein homologues in native tissues, polyclonal antibodies specific for mouse Rhbg were developed and tested on Western blots. As
shown in Fig. 10B, Rhbg is specifically expressed in liver
and kidney but not heart, consistent with the results of RNA analysis (Figs. 6B and 7). The native Rhbg protein was estimated to
have an apparent molecular mass of 50-55 kDa. It was of similar size in both the liver and kidney forms (Fig. 10B, lanes
3 and 5), but was, as expected, slightly smaller than
the stably expressed RhBG having C-terminal tags (Fig. 10A,
lanes 3 and 4). PNGase F treatment deglycosylated
Rhbg (Fig. 10B, lanes 4 and 6),
resulting in a product that had a size similar to the in
vitro translated unglycosylated RhBG (Fig. 9A,
lane 6). Taken together, these results provide evidence for
49NHS51 and 46NHS48 to
be the most probable attachment site of N-linked glycan on the exoloop 1 of RhBG and Rhbg, respectively.
We have identified human RHBG and mouse Rhbg
as a novel pair of genes encoding two polytopic membrane proteins that
are homologous to erythroid and nonerythroid Rh glycoproteins.
Molecular cloning revealed that the translated sequences of
RHBG and Rhbg bear highest similarity in both
primary and secondary structures among all Rh homologues known to date.
RNA blot and in situ hybridization determined the pattern of
RhBG/Rhbg expression in specific nonerythroid tissues. Biochemical
characterization and confocal imaging analysis established RhBG and
Rhbg to be N-glycosylated proteins that mainly reside in the
cell plasma membrane. The shared conservation in structural features,
in chromosomal synteny, and in tissue distribution defines RhBG and
Rhbg as new members of the Rh superfamily; it also pinpoints
RHBG and Rhbg as an orthologous gene group that most probably specify the same functional role(s) in mammalian species.
Furthermore, parallel analyses of Rh homologues and numerous known Amt
sequences have, for the first time, placed the entire Rh family as a
unique gene cluster within the Amt superfamily. Collectively, these
results lead to new testable hypotheses as to the function of Rh,
suggesting that the RhBG protein homologues may be the long sought
after major ammonium transporters in mammals.
The RhBG or Rhbg full-length cDNA predicted a polypeptide sequence
of 458 or 455 amino acids. Like the erythroid (18-20, 22) and
nonerythroid homologues (26), the first AUG codon of neither RHBG nor Rhbg mRNA resides in the typical
Kozak context (35); nevertheless, several lines of evidence support its
assignment as the genuine initiation signal for RhBG or Rhbg
translation. 1) There are stop signals but no in-frame ATG triplets
upstream of the Comparison of RhBG/Rhbg with other known homologues provides insights
into the protein structure and molecular evolutionary genetics of the
entire family. The Rh protein homologues from diverse organisms have
been subdivided into three interrelated groups (23), which may or may
not carry N-linked glycans. The primitive group consists of
homologues from unicellular slime molds, multicellular protozoans, and
metazoans (nematode and arthropods) that lack RBC or such formed organs
as liver and kidneys. The biological function(s) of these Rh homologues
is still unknown. The erythroid group includes only members homologous
to human RhAG and RhCE/D, which coexist in RBC of all mammals (40, 41). Here, we show that RhBG/Rhbg is more similar to RhCG/Rhcg (26) than to
the RBC Rh proteins (18-20) at the level of primary and secondary
structures. This similarity, with the observed tissue distribution,
clearly delineates RhBG and Rhbg as novel members of the expanding
nonerythroid group. Despite its separate chromosomal location and
unique C-terminal segment, RhBG/Rhbg resembles other members of the
family by having a highly conserved 12-TM fold. The shared TM fold is a
signature characterized by an invariant packing of internal TM2-11
segments, including the conserved positioning of membrane-embedded
D/E-negative charges. This topological structure may define a transport
function, as it is similar to a large repertoire of transporters that
act as either antiporters or symporters that lack an ATP-binding
cassette (42).
RhAG and RhCE/D homologues are coexpressed in and largely restricted to
erythroid cell lineages in both mice and humans (18-20, 22). This
coordinate has been hypothesized to stipulate assembly of the Rh
multisubunit complex required for specific functional adaptation in the
RBC membrane (43, 44). In contrast, the nonerythroid homologues often
have a much broader spectrum of tissue distribution. Although both are
expressed in the kidney, Rhbg and Rhcg are clearly localized to
discrete regions of the organ and are not overlapping in other complex
tissues. In brief, Rhbg is likely expressed in the convoluted tubules
and Henle's loops, whereas Rhcg is mainly concentrated in the
collecting tubules (26). Furthermore, RhBG/Rhbg is expressed in liver,
skin, and ovary, but RhCG/Rhcg is expressed highly in the testis
seminiferous tubules and moderately in several other tissues, namely,
brain, pancreas, and prostate (26). These spatial differences,
including a mutual exclusive expression in the primary sex organs
(ovary and testis), suggest that RhBG/Rhbg and RhCG/Rhcg may fulfill specific functional role(s) in those tissues. Because RhBG and RhCG can
reach the plasma membrane of heterologous cells, their tissue
specificity may be determined largely by control of their discrete
promoters at the level of transcription. The gaining of such regulatory
novelties was likely driven by a translocation event predating
mammalian radiation, given that Rhbg/RHBG and Rhcg/RHCG are highly homologous yet localized to the
different chromosomes of mice and humans.
As determined by FISH mapping, the human RHBG gene resides
at 1q21.3 of chromosome 1. Such a map location falls into the
chromosomal region recently shown to contain candidate genes for one
form of autosomal dominant medullary cystic kidney disease (38). The
disease complex consists of a genetically heterogeneous group and is
adult onset, manifesting renal cyst formation in the medulla or
the corticomedullary junction accompanying salt wasting (45). The
specific expression of RhBG in the kidney, combined with its locus map
and possible transport activity, suggests RHBG as a candidate gene for the disease association. A defective exchange of
NH4+ with other ions may lead to salt
wasting and ultimately the pathological state. Hence, identification of
possible mutations in the RHBG gene in the afflicted
families may provide significant insight into the protein function.
Amt represents a category of proteins not yet described in vertebrates,
including mammals, despite their presence in such low order animals as
nematodes (13). The Rh proteins may act as membrane transporters
participating in homeostatic preservation in many organisms, given
their structural and topological conservation and wide distribution in
slime molds to humans (23). A similarity search has linked Amt to human
RBC Rh proteins (17). We define the entire Rh family, including
Rhbg/RhBG, as a single phylogenetic group that occurs in
Eucarya only but falls into the Amt superfamily. Although
future experimental work will be required to prove Rh to be actual
NH4+ transporters, this clustering
unveils new information about the evolution, structure, and function of
the two families.
Concerning the evolution relationship, our finding that the Rh cluster
joins two Amt clusters provides evidence that the Rh family is derived
from NH4+ transporter ancestors. Indeed,
extensive search shows that no expressed sequence tags from the human
genome other than those of Rh are more homologous to Amt members. It is
worth noting that Amt members of Cluster II are distributed in diverse
organisms from three life domains, whereas Clusters I and III are
relatively homogeneous in organism classification. The current rooting
indicates a late origin and duplication of Rh precursor genes from
Cluster II ancestors. This might cope with evolution from unicellular to multicellular organisms, because Rh is absent in bacteria and yeast
but present in D. discoideum (23), a unicellular slime mold
with a multicellular developmental program (46). Structurally, members
of the Rh family share sequence homology with that of the Amt
superfamily in a composite fashion and with some degrees of variation.
For example, RhBG/Rhbg is most similar to the cyanobacterial and (to a
lesser extent) archaeal Amt proteins that are members present in the
Cluster II subfamily. This observation, together with their expression
in the liver, kidney, and skin, points toward RhBG and its orthologues
possibly as the major NH4+ transporters
in mammalians. Nonetheless, significant divergence exists between the
Rh and Amt families. The noted structural changes may underlie a
functional transition from NH4+
assimilation to NH4+ disposal, given the
differences in the uptake of required nutrients and in the external and
internal milieu between microorganisms and animals.
The Rh homologue occurs as a single-copy gene in the slime mold and
fruit fly but in the form of multiple copies in mammalian species (23).
With the identification of Rhbg and RHBG, four and five gene paralogues are now known to reside on four and three different chromosomes in the mouse and human genomes, respectively. This type of expansion of the Rh family during mammalian evolution implies two possible outcomes with regard to functional specification. 1) If they serve to transport the same or similar ligand(s)
(e.g. NH4+ or its
derivatives), the Rh paralogues may differ in kinetics and regulatory
modes to meet the physiological requirements of the target cells or
organs. A noted example as such is the duplication and expression of
various homologues for urea transport in ureotelic animals (47). 2)
Conversely the multiple Rh homologues may each perform a completely
different function, say, each transporting a structurally unrelated
ligand. We favor the first hypothesis (i.e. the Rh protein
homologues perform some similar related functions) taking into
consideration the remarkable evolutionary conservation of the entire Rh
family in both the primary and secondary structures. Nevertheless, the
possibility that Rh proteins may have additional associated functional
roles cannot be excluded, given that yeast MEP2 not only acts as an
NH4+ permease but also regulates
pseudohyphal differentiation (48). There is reason to believe that the
function of Rh as transporters is a complex biochemical process,
including possible coupling with energy or some ion potentials, such as
the K+/Na+/Cl
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin cDNA
probe was used as a control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (148K):
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Fig. 1.
RhBG and Rhbg: sequence and alignment with
erythroid RhAG/Rhag and nonerythroid RhCG/Rhcg. Sequences
are aligned by means of MegAlign, and their numbering is indicated at
the far right. Identical amino acid (n 4) is shaded
in yellow and similar ones (n = 4) in
gray or red. The insertion of
8AAG10 in RhBG is indicated (dots).
NX(S/T) motif (bar), D/E conservation (red
star) in TM4 or -5, and E/Q change (blue star) in TM1
or -5 are shown. GenBankTM accession numbers: RhBG, AF193807; Rhbg,
AF193808; RhAG, AF031548; Rhag, AF057526; RhCG, AF193809; Rhcg,
AF193810.
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Fig. 2.
Hydropathy profile and secondary structure of
RhBG: comparison with RhAG and RhCG. The mouse counterpart of each
protein is not shown, because it shows an apparently identical pattern.
Analysis was carried out using the Kyte-Doolittle scale and Chou-Fasman
algorithm in Protean software. The hydrophobic segments as putative TM
domains are indicated in Roman numerals. Secondary structure
symbols are: A, -helix, B,
-sheet, and
T, turns.
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[in a new window]
Fig. 3.
Relatedness of the Rh and Amt families by
protein sequence clustering. Left, the dendrogram of
the relationships between the Rh and Amt proteins constructed with the
ClustalW program. Four major clusters, including Rh are recognized:
Cluster I is subdivided into class Ia and Ib, whereas the Rh cluster
intercepts Amt Clusters I and II. CG6499 is a putative Amt from fruit
fly. Note that some branch points within individual clusters remain
ambiguous. The right panel shows accession, organism, and
structure parameters of each protein. Human RhBG (boxed) is
used as a reference (100%) for computation of sequence identity
(ID) and overall similarity (SM) using the
J. Hein procedure. TM numbering is based on either data base
search or Kyte-Doolittle hydropathy analysis.
View larger version (114K):
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Fig. 4.
Sequence homology noted between human RhBG
and two Amt members from subfamily II. Sequences are aligned by
means of MegAlign with minor manual manipulations; their designations
are: Hs.RhBG, human RhBG; Sp.AmtA,
Synechococcus PCC7002 ammonium transporter A (AAF21444, no.
49 in Fig. 3); Sy.Amt1, Synechocystis sp.
ammonium transporter 1 (P72935, no. 51 in Fig. 3); and
Rh.maj, the majority consensus derived from a collective
alignment of mammalian Rh glycoprotein homologues. Amino acid number is
denoted at the far right. Identical residues are colored in
blue and similar ones in yellow. A/G or G/A
substitutions are denoted in red letters and shaded in
gray. Gaps are indicated with dashes. In Rh.maj,
nonconserved amino acid positions are marked with crosses.
Note the composite identity and similarity RhBG shares with either
Sp.AmtA or Sy.Amt1.
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Fig. 5.
Chromosomal mapping of human RHBG
and mouse Rhbg genes. A, diagram
for the location of RHBG at 1q21.3. RHBG BAC DNA
was used as FISH probes to paint interphase chromosomes. B,
diagram for the linkage map of mouse Rhbg on chromosome 3 (the centromere toward the top). A 3-centimorgan scale bar
is shown at the right. Loci mappings to the same position are listed in
alphabetical order. Corresponding human map positions for
underlined loci are listed to the left.
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Fig. 6.
MTN (multiple tissue Northern) blot
analysis. A, RHBG expression in human adult
and fetal tissues. B, Rhbg expression in mouse
adult tissues and at embryonic gestational stages. 2 µg of poly (A)+
RNA was loaded to each lane. Size markers are indicated at left.
Tissues are designated above each panel: s.muscle, skeletal
muscle; s.intestine, small intestine, and WBC,
white blood cells. Three bands of varying intensity on human MTN blots
are indicated by arrows, but only one major band is seen on
mouse blots. Actin cDNA hybridization was relatively constant (not
shown).
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Fig. 7.
Rhbg expression in mouse embryos and adult
tissues. A, Rhbg expression in the 16.5-day
mouse embryo: skin (S) and liver (L). Note the
kidney is not seen in this section. B and C, low
and high magnification of Rhbg expression in skin
(H, hair; HF, hair follicle; P,
papilla). D, Rhbg signal in adult mouse kidney
(C, cortex; M, medulla). E, high
magnification of Rhbg expression in the kidney
(CT, convoluted tubules, indicated by arrows;
RC, renal capsules). F, image of E. G,
Rhbg expression in the liver. H, high
magnification of Rhbg expression in liver (HC, hepatocyte,
arrows; CV, central vein). I, image of
H. A, D, F, G, and
I represent dark fields. B, C,
E, and H represent bright fields.
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Fig. 8.
Localization of RhBG to the plasma membrane
by confocal microscopy. Cultured cells were transfected with
RHBG-GFP (in pEGFP-N3) or GFP-RHBG plasmid (in
pEGFP-C1). Images were collected on a Bio-Rad MRC confocal
laser-scanning microscope. A, B, and
C, homologous HepG2 cells. D, E, and
F, homologous HEK293 cells. G, H, and
I, heterologous HeLa cells. A, D, and
G, controls (pEGFP-N3 vector). B, E,
and H, RHBG-GFP fusion. C,
F, and I, GFP-RHBG fusion.
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Fig. 9.
In vitro protein translation and
processing of human RhBG. A, 12% SDS-PAGE of in
vitro translated RhBG in the absence of CPMM. Positive
(Pos) and negative (Neg) controls: Pos
1, yeast -mating factor; Pos 2, luciferase;
Neg 1, no template; Neg 2, pYES2 alone.
RhBG, pYES2/RhBG; RhBG-myc,
pcDNA3.1/RhBG-myc. B, 12% SDS-PAGE of in
vitro translated RhBG in the presence of CPMM. Pos 1,
yeast
-mating factor; Neg 1, no template. RhBG and
RhBG-myc are as in A.
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Fig. 10.
Western blot analysis of human RhBG from
stable HEK293 cells and mouse Rhbg from native tissues.
A, plasma membranes of HEK293 cells stably expressing the
tagged human RhBG were separated by 12% SDS-PAGE and blotted. RhBG was
visualized with RhBG C tail-specific antisera (left panel)
or anti-Myc monoclonal (right panel). Human RBC ghost
membranes were used as controls. +, DTT or PNGase F added; , DTT or
PNGase F not added. B, Western blot of mouse Rhbg. Membrane
proteins from mouse heart, liver, and kidney were fractionated,
blotted, and probed with anti-Rhbg polyclonal antibodies raised with
synthetic peptides. + and
, PNGase F added and not added. Size
markers (in kDa) are shown at the left margin. Dilution of
antibodies is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
38A major transcription start site in the
5'-promoter of the RHBG gene (see AF219977). 2) The sizes of
human RhBG and mouse Rhbg proteins appear identical, regardless of
whether they were produced in vitro or derived from stable
cells or native tissues. 3) Consistent with the mRNA distribution,
the specific expression of mouse Rhbg protein was confirmed by Western
blots using antibodies raised against the deduced peptide sequences. 4)
Most significantly, the assigned translation initiator is the only
methionine N-terminal to the NHS sequon on exoloop 1 that was
evidently glycosylated in RhBG and Rhbg proteins.
cotransport
systems. The recognition of RhBG/Rhbg as Cluster II Amt homologues
should lead to a better understanding of the structure-function
relationships of the entire Rh family concerning ammonium transport in
ammonotelic, urecotelic, and ureotelic organisms.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Drs. Olga O. Blumenfeld and Narla Mohandas for critical comments on the manuscript. We thank Michael Cammer (The Albert Einstein College of Medicine) for confocal imaging analysis, Jan-Fang Cheng (The Lawrence Berkeley Laboratory) for screening BAC genomic clones, and Mary Barter (The Jackson Laboratory) for providing Fig. 5B. We appreciate Ying Chen for technical help and staff of the Microchemistry Laboratory for DNA sequencing.
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FOOTNOTES |
---|
* This work was supported by Institutional Fund D33210101 of the New York Blood Center and in part by National Institutes of Health Grant HL54459 (to C.-H.H).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF193807 and AF193808.
¶ To whom correspondence should be addressed: Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Ctr., 310 East 67th St., New York, NY 10021. Tel.: 212-570-3388; Fax: 212-570-3251; E-mail: chuang@nybc.org.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M007528200
2 Z. Liu, Y. Chen, J.-F. Cheng, and C.-H. Huang, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: Amt, ammonium transporter(s); RBC, red cell(s); TM, transmembrane; GSP, gene-specific primer(s); RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; GFP, green fluorescence protein; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; PAGE, polyacrylamide gel electrophoresis; UTR, untranslated region; bp, base pair(s); E10, exon 10; CPMM, canine pancreatic microsomal membrane.
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