Glycophorins C and D are generated by the use of... translation initiation sites [letter]

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1996 88: 2364-2365
Glycophorins C and D are generated by the use of alternative
translation initiation sites [letter]
C Le Van Kim, V Piller, JP Cartron and Y Colin
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2364
CORRESPONDENCE
Glycophorins C and
D Are Generated by the Use of Alternative Translation Initiation Sites
To the Editor:
Human glycophorin C (GPC) and glycophorin D (GPD) are two
membrane sialoglycoproteins that cany the Gerbich (Ge) blood
group antigens. Although the function of these glycoproteins in a
wide variety of cells has not been fully elucidated,' analysis of red
blood cell variants indicated that GPC and GPD, by interacting
with the palmitoylated erythrocyte membrane protein p55 and the
cytoskeleton protein 4.1, mightplay a pivotal role in regulating
the mechanical stability andthe deformability ofthe erythrocyte
membrane. Indeed, the simultaneous lack of GPC and GPD at the
red blood cell surface of Ge-negative variants of the Leach type is
associated with elliptocytosis, absence of p55, and a 15% to 20%
decreased protein 4.1 level.' The entire primary structure of GPC
(128 amino acids) was determined both by cDNA and amino acid
sequencing, but the NH2 terminus of GPD could not be determined
because it was found to be blocked. However, partial sequencing of
GPD together with immunologic studies indicated that GPD is an
abridged form of GPC in the NH2 terminal part.' Furthermore. we
have shown that GPC and GPD are encoded by a unique ubiquitous
gene, GYPC, whose expression is activated in erythroid cells.' We
describe here the molecular mechanism that accounts for the synthesis of both GPC and GPD by the same gene.
Several mechanisms could account for the production of GPC and
GPD by the GYPC gene: ( l ) alternative splicing ofthe primary
transcript and/or use oftwo distinct promoters, (2) posttranslationnal
processing of GPC, and (3) alternative initiation of translation at
two in phase AUGs of a unique mRNA. There was no evidence
from Northern blot and primer extension analysis for the existence
of distinct transcripts encoding GPC and GPD! In contrast. examination of the GPC cDNA sequence indicated that GPC and GPD might
be produced from the same mRNA by alternative initiation at the
AUG codons 1 and22, respectively (position 1 indicates thefirst
ATG initiating the GPC protein), according to the leaky scanning
mechanism permitting ribosomes to initiate translation either at the
first or at the second internal AUG.' Indeed, the nucleotides surrounding theATG codon for Met-l (5"CCAGGA ATG T-3') do
not fit properlywith the consensus translation initiation sequence
( 5 ' 4 CCNGCC ATG G-3') found in viral and eukaryotic mRNAs.6
However, the ATG codon for Met-22 (5' CCGGGG ATG G) is
A
CCAGGAAfG 1
CCGGGG ATG G
pGPC
Mel-l
CMV
Mcl-22
promoter
CCGGGG ATG G
*~CA.ATGTOI
I
f
1
MBI-22
CCAGGA ATG T
CCGGGG ACG G
PGPCNCATGZZ
Mm-l
CCAGGA ACG T
CCGGGG ACG G
*PCmut.ATGlr22
Thr-l
CCAGGA ATG G
Thr-21
CCGGGG ATG G
*PCm~I+Q
Met-l
Mel-22
b
Fig 1. Expression of GPC/GPD-related polypeptides by transfected COS-7 cells. (A) Schematic representation ofexpression plasmids carrying intact or mutated forms of the GPC cDNA. Only relevant sequences arround AUGs 1 and 22 are indicated. Mutations
were introduced by M13 site-directed
mutagenesis (Sculptor kit; Amersham, Bucks, UKI. cDNAs were then subcloned in the pcDNAl expression vector (Invitrogen, San Diego, CAI. (B)Immunoprecipitation
of GPC- andlor GPD-related polypeptides by the
anti-GPCIGPD L857
rabbit polyclonal antibody. COS-7 cells were transfected by electroporation. Fortyeight hours aftertransfection, cells were labeled for
30 minutes with "S-methionine (200 pCiI2 x l o 6cells), chased for 3
hours with cold methionine (1.25 mmolIL final concentration), and
immunoprecipitated withthe L857 polyclonalantibodydirected
against the common C-terminal part
of GPC and GPD polypeptides.'
Immunoprecipitates were resolved by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and shown by autoradiography.
Recombinant plasmids used for transfection were pcDNAl vector
alone, lane 1; pGPC, lanes 2 and 6; pGPCaArG1, lane 3;pGPCmA
.,TG,
l u,
lane 5; and pGP,C
,,,,
lane 7. Lane 8. GPC
lane 4; pGPC,.ArG+
membranes.'
and GPD extracted froml'25radiolabeled red blood cell
B
1 2 3 4 5 6 7 8
r
k Da
91.4
69
--
46
-
30
"
21.5
-
14.3
-.
4
GPC
4
GPD
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CORRESPONDENCE
present in the optimal context, with a purine at position -3 from
the initiator ATG and a guanine at position +4 (Fig IA). The use
of the second AUG would result in the translation of a polypeptide
chain of 107 amino acid residues having the immunologic properties
and the expected size for GPD, assuming the presence of only six
0-glycosidically linked tetrasaccharide chains.
To test this hypothesis, intact or in vitro mutated forms of the
GPC cDNA were subcloned in the pCDNAl expression vector and
used for transfection of COS-7 cells. Recombinant polypeptides were
immunoprecipitated with the L857 polyclonal antibody directed
against the common C-terminal end of GPC and GPD3 (Fig IB).
Lanes 2 and 6 indicated that the pGPC plasmid carrying intact AUG
codons 1 and 22 could direct the synthesis of GPC- and GPDrelated polypeptides with the same size (39 and 29 k D , respectively),
immunologic properties, and relative levels as the GPC and GPD
glycoproteins extracted from red blood cell membranes (lane 8). A
T to G mutation at nucleotide +4 (plasmid pGPC,,+,), restoring a
more consensus translation initation motif around thefirst AUG.
resulted in an a twofold overexpression of GPC as compared with
GPD (lane 7). pGPCA.ATGI,
in which the first ATG has been deleted,
led to the synthesis of only the GPD related polypeptide (lane 3),
whereas the ATG22ACG mutation (pGPC,,,.ATG22)resulted in the
synthesis of GPC only (iane 4), excluding the possibility that GPD
should arise from proteolytic cleavage of GPC. Neither GPC nor
GPD was synthesized from pGPCml.ATGI+ZZ
(lane 5 ) , in which both
ATG-l and -22 have been substituted by an ACG codon. The 26.5and 25.5-kD species detected in the absence of GPC and GPD (lane
5) and in less proportion in the absence of only GPC (lane 3) or
GPD (lane 4) most likely resulted from the use of downstream
initiation codons, suggesting that the GYPC gene should constitute
a very permissive model for the initiation of eukaryotic translation.
In conclusion, our transfection and mutagenesis experiments
showed that a unique cDNA derived from the GYPC gene was able
to direct the synthesis of both GPC- and GPD-related polypeptides.
Thus, these two proteins represent anew example of analogous
2365
eukaryotic polypeptides arising from the same mRNA by the alternative use of two in phase AUGs initiator codons.
ACKNOWLEDGMENT
We thank Magali Clerget for technical assistance.
Caroline Le Van Kim
Veronique Piller
Jean-Pierre Cartron
Yves Colin
INSERM U76
Institut National de la Transfusion Sanguine
Paris, France
REFERENCES
1. Colin Y,
Le VanKim C: Gerbich blood groups and minor
glycophorins, in Cartron JP, Rouger P (eds): Blood Cell Biochemistry, v01 6. New York, NY, Plenum, 1995, p 331
2. Alloisio N. Dalla Venezia N, Rana A, Andrabi K, Texier P,
Gilsanz F, Cartron JP, Delaunay J, Chishti AH: Evidence that red
blood cell protein p55 may participate inthe skeleton-membrane
linkage that involves protein 4.1 and glycophorin C. Blood 82:1323,
1993
3. El-Maliki B, Blanchard D, Dahr W, Beyreuther K, Camon
JP: Structural homology between glycophorins C and D of human
erythrocytes. Eur J Biochem 183539, 1989
4. Le Van Kim C, Colin Y , Mitjavila MT, Clerget M, Dubart A,
Nakazawa M, Vainchenker W, Cartron JP: Structure of the promoter
region and tissue specificity of the human glycophorin C. J Biol
Chem 264:20407, 1989
5. Kozak M: The scanning model for translation: An update. J
Cell Biol 108:229, 1989
6. KO& M Atleast six nucleotidesprecedingtheAUGinitiator
codon enhance translationin mammalian cells.J Mol Biol1%947, 1987