(Received for publication, August 29, 1994; and in revised form, November 14, 1994)
From the
Alternative splicing of the primary transcript for human complement protein C2 generates templates for translation of a secreted (C2 long) protein and an intracellular (C2 short) form in liver, bronchoalveolar macrophages, and fibroblasts. The approximate ratio of C2 long to C2 short mRNA is 2:1. The C2 short mRNA does not contain the 396-base pair encompassed by exons 2 and 3 of the full-length C2 long and thus lacks codons for the 5 carboxyl-terminal residues of the signal peptide. Synthesis of C2 in cells transfected with full-length RNA corresponding to each of the transcripts show that C2 long is secreted within a half-time of approximately 1 h and that C2 short is not secreted. Cell-free biosynthesis in the presence of microsomes demonstrate that this intracellular C2 protein (70 kDa) is apparently capable of traversing the membrane of the endoplasmic reticulum. Though the function of the intracellular C2 protein is unknown, it is abundant in all cell types that express the C2 gene.
The second component of human complement (C2) is a single chain
glycoprotein (molecular mass, 100 kDa) (1, 2) that carries the serine protease domain of a
bimolecular complex enzyme comprised of cleavage products of C2 (C2a)
and the fourth (C4) complement protein (C4b). This enzyme is
responsible for cleavage of complement protein C3 to its biologically
active fragments. C2 is encoded by a single gene that is on human
chromosome 6p within the class III region of the major
histocompatibility complex(3) . The C2 protein is synthesized
in liver hepatocytes (4, 5) and in several other cell
types in extrahepatic
tissues(6, 7, 8, 9, 10) .
Many years ago, three forms of C2 (84, 79, and 70 kDa) were detected
in cell lysates of a metabolically labeled well differentiated human
hepatoma cell line (HepG2)(5) . Initial data indicated that
each was derived from a separate primary translation product, that each
is glycosylated, but that only the 84-kDa C2 polypeptide is secreted
(half-time 1 h). The other two C2 polypeptides remained
cell-associated throughout the observation period (>6 h).
Subsequently, these observations were replicated in studies of every
cell type that expresses C2 protein(11, 12) , but the
origin of the isoforms and the cellular compartment(s) in which the 79-
and 70-kDa C2 proteins reside were unknown. Recently, in a study of C2
deficiency type I, we noted that a reverse transcriptase polymerase
chain reaction (PCR) (
)that should have generated a 786-bp
fragment from the 5` end of both normal and C2-deficient mRNA instead
generated two major bands, one of the predicted size and another that
was about 400 bp (see Fig. 1in (13) ). The
reproducibility and abundance of this
400-bp PCR product suggested
the possibility that a C2 mRNA not previously recognized was the
template for one of the cell-associated C2 proteins. In 1994, Cheng and
Volanakis also found multiple C2 transcripts by PCR amplification of
mRNA from HepG2, normal liver, and two other cell lines(14) .
They speculated that one or several of these transcripts, if
translated, would give rise to variant C2 proteins; among them the
previously recognized cell-associated C2 isoforms. The current study
was undertaken to test that hypothesis.
Figure 1:
C2
gene structure and alternatively spliced mRNA isoforms. 18 exons and
introns of human C2 are drawn approximately to scale. Exons 1-10 are shown with the number of nucleotides indicated
within each exon. The two BamHI (B) and HindIII (H) sites were engineered in the
oligonucleotides a, d, and b, respectively.
The C2 long 748-bp and C2 short 352-bp fragments were sequenced.
oligonucleotides (c) used to prime reverse
transcriptase; (a + b) used to generate the cDNA
fragment for analysis; (d + b) used to generate
the 5` end of full-length C2 long and short.
3` end of exon 1
that encodes the signal peptide (not drawn to scale).
GLADS, portion of signal peptide encoded within exon
2.
Figure 3: A, Nuclease protection of C2 mRNA in normal human liver (lane4) and HepG2 (lane5). Lanes1 and 2, undigested probe; lane3 tRNA. B, antisense riboprobe used for nuclease protection.
Mouse fibroblast
L-cells were grown to 70% confluence in 24-well plates containing
350 µl of Dulbecco's modified Eagle's medium and 10%
bovine calf serum. Transfection of the L-cells was accomplished with 4
µg of DNA/well using the calcium phosphate precipitation method
exactly as described(21) . Thirty-six hours following
transfection, the cells were pulse-labeled for 30 min with 250
µCi/ml of [
S]methionine (specific activity,
1,000 Ci/mmol from ICN Biomedicals, Irvine, CA) in
Dulbecco's modified Eagle's medium lacking methionine in
the presence of 10% (v/v) dialyzed fetal calf serum and then chased for
30 min, 1, 2, 4, and up to 24 h. At each time point, the medium was
collected, and the cells were lysed as described(5) . Aliquots
were assayed for total protein synthesis by trichloroacetic acid
precipitation, and the balance was used to precipitate human C2 with
sheep antiserum (Miles Scientific, Naperville, IL) exactly as
described(22) . Immune complexes were collected with excess
protein A, washed, released by boiling in sample buffer, and applied to
8% SDS-polyacrylamide gel electrophoresis under reducing
conditions(23) . After electrophoresis, gels were stained in
Coomassie Brilliant Blue, destained, and dried for fluorography.
Figure 2: PCR fragments of 786 (C2 long) and 390 bp (C2 short) generated from mRNA in normal human liver, HepG2, fibroblasts from normal, C2 deficient type I, C2 deficient type II and bronchoalveolar lavage cells. tRNA served as negative control.
Digestion of the 786- and 390-bp fragments with BamHI and HindIII (sites engineered by primers a and b) generated 748- and 352-bp fragments, respectively, which were subcloned separately and sequenced. The results represented in Fig. 1show that the 352-bp fragment completely lacked the sequence corresponding to exons 2 and 3. This predicted a translation product that would lack the carboxyl-terminal 5 amino acids of the peptide leader sequence (encoded by the proximal 15 bp of exon 2). Since both exon 2 (210 bp) and exon 3 (186 bp) are in phase, the transcript with this segment deleted could theoretically serve as template for a C2 protein shorter by the 132 amino acids that are encoded by these two exons.
The results of four separate experiments showed that the C2 short mRNA was present in HepG2 and normal liver at about one-half of the concentration of C2 long.
Figure 4:
Kinetics of synthesis and secretion of C2
short and C2 long. A, synthesis and secretion of C2-short
protein in transfected L-cells. Transfectants were labeled for 30 min
with [S]methionine and chased with unlabeled
methionine for intervals up to 24 h. At timed intervals, culture media
were harvested and cells were solubilized, immunoprecipitated, and
analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. Lanes1-6 contain cell lysates, and lanes7-12 contain extracellular media from chase time
points 0, 0.5, 1, 2, 4, and 24 h. Intracellular C2-short protein
(molecular mass,
69 kDa) but not extracellular C2 protein is
identified even on a long exposure (3
) film (data not shown). B, kinetics of synthesis and secretion of C2 long protein in
transfected L-cells.
84- (intracellular C2 short) and
92-kDa
(secreted form) C2 polypeptides are identified. Lanes are labeled
exactly as in A.
Figure 5: Indirect immunofluorescence in the L-cells transfected with C2 short and C2 long. C2 short transfectants stain with the monoclonal anti-human C2 antibody strongly in the perinuclear regions (panelA). The pattern of C2 long transfectants was much less intense and more diffuse (panelB). PanelsC (C2 short) and D (C2 long) are monoclonal control antibodies. Exposure time was 4 s each.
Figure 6:
In
vitro translation of C2-short and C2-long C2-short (A) or C2 long
(B) mRNA translated in a rabbit reticulocyte lysate system supplemented
with 1 mCi/ml [S]methionine in the presence or
absence of dog pancreas microsomes. After translation was completed,
aliquots of the translation reactions were treated with a mixture of
trypsin and chymotrypsin in the presence or absence of 1% Triton X-100.
C2 short is translocated into the microsomes. PanelA, arrows show primary translation product
62 kDa and translocated polypeptide
67 kDa; panelB, arrows show 77 and 82 kDa,
respectively.
Alternative splicing of precursor mRNA is but one of several mechanisms generating control of gene expression at the level of translation(26) . In addition, alternative splicing can govern the destination, size, and function of the protein products derived from a single gene(26) . Abundant evidence has been obtained for alternative initiation and alternative splicing events in the transcription and processing of human (5, 10, 14, 27) and murine (28, 29, 30, 31) mRNA derived from the homologous major histocompatibility complex-linked complement C2 and factor B genes. For example, both murine factor B (30) and human C2 (27) mRNA forms expressed in a tissue-specific pattern vary in length of 5`-untranslated regions. In the case of murine factor B, one of the initiation codons within this 5` extension has an effect on the rate of translation of factor B protein(32) .
The cell biological implications have not been ascertained for the several alternatively spliced mRNA species identified in murine and human tissues expressing C2.
In the present report, we provide evidence
that a relatively abundant transcript lacking exons 2 and 3 (ex 2,
3) is the template for a previously recognized
70-kDa C2 protein
that is found in lysates of every cell type that synthesizes and
secretes native C2. This conclusion is based on (a) reverse
transcriptase PCR identification of a truncated C2 mRNA in several
different tissues, (b) quantitation of the truncated C2 by
nuclease protection that shows a 1:2 relative abundance compared to
full-length C2 mRNA, (c) sequence analysis that shows deletion
of exons 2 and 3 at the authentic splice junctions, (d)
expression of the truncated isoform in murine L-cells generates a
70-kDa polypeptide in cell lysates but no C2 protein is secreted into
the medium. The subcellular location of the product of this truncated
C2 mRNA is intracellular as suggested by fluorescent antibody staining
of transfected L-cells. No cell surface membrane staining was apparent.
Studies of cell-free synthesis in the presence of microsomes clearly
establish that the truncated C2 protein can traverse the endoplasmic
reticulum membrane even though it lacks 5 amino acids at the carboxyl
terminus of the leader peptide. This deletion would likely make the
product resistant to cleavage by the signal peptidase. This C2 protein
is also lacking a portion of the amino-terminal domain critical for
optimal binding to the C4b protein, which is required for generating
the C3 cleaving enzyme of the classical activation
pathway(33) .
The present study does not address the
biological function of the 70-kDa intracellular C2 protein. It is
possible that the 70-kDa C2 protein has no function and/or is simply a
vestige of an ancestral protein. On the other hand, its presence in
relatively high abundance within cells that express C2 and its
regulation by interferon- (34) suggest that it may play a
role in the intracellular traffic of C2 itself or of proteins that can
interact with C2. Since the cleavage site that activates the C2 serine
proteinase is intact, it is possible that it may serve an enzymatic
function within the cell.