(Received for publication, May 8, 1995; and in revised form, July 20, 1995)
From the
Laminin-5 is a heterotrimer composed of 3,
3, and
2 chains, produced by keratinocytes and the human squamous cell
carcinoma line (SCC-25), and is one of the candidate proteins for the
genetic lesion in junctional epidermolysis bullosa. Two-dimensional
SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing
conditions; second dimension, reducing conditions) revealed that the
immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of
3,
3, and
2 monomers, a
3
2 heterodimer, and an
3
3
2 heterotrimer. The presence of the
3
2
heterodimer, but not heterodimers containing an
3 chain and any of
the other chains, was suggestive of assembly of laminin-5 proceeding
from a
3
2 heterodimer to an
3
3
2 heterotrimer.
We showed, by cotransfection experiments using full-length recombinant
3 and
2 chains in a human cell line devoid of endogenous
laminin-5, that stable heterodimers can be formed in the absence of
3 chain expression. In the SCC-25 cell fraction, the
3
monomer pool was the smallest of the monomers. Pulse-chase experiments
using the cell fraction also indicated that the heterotrimer was
assembled after a 10-min pulse and was nearly absent after a 24-h
chase. These results are consistent with the synthesis of
3 being
limiting for heterotrimer assembly, with rapid association of the
3 chain with
3
2 heterodimers to form complete
heterotrimers. Treatment with tunicamycin reduced the size of each of
the laminin-5 subunits, indicating that all chains are glycosylated,
but that N-linked glycosylation is not necessary for chain
assembly and secretion.
Laminin-5 (kalinin/nicein) is an epithelium-specific laminin
subtype and is a component of the anchoring filament of the lamina
lucida in the basement membrane of the skin(1) . The anchoring
filament is the bridge between hemidesmosomes and the lamina densa
region of the basement membrane and is believed to mediate the adhesion
of the epithelium to the basement membrane. Laminin-5 is therefore one
of the primary adhesion proteins holding together the epidermis and the
dermis. Laminin-5 is initially synthesized in a cell-associated form,
estimated to be 460 kDa, and is composed of three polypeptides: the
200-kDa (3), 145-kDa (
3), and 155-kDa (
2)
chains(2) . The three chains are presumed to form a cruciform
structure where the chains are bound by disulfide linkages(2) .
The two heterotrimeric forms in keratinocyte cell-conditioned culture
medium are derived from the cellular form by extracellular processing.
A 440-kDa medium form results from the processing of the 200-kDa
3
chain to 165 kDa, while the 400-kDa form is derived from the 440-kDa
form by extracellular processing of the 155-kDa
2 chain to 105
kDa(2) .
A number of variant laminin subunits, in addition
to those of laminin-5, are assembled in the rough endoplasmic
reticulum. N-Linked oligosaccharide processing of some of
these has been shown to occur within the Golgi apparatus prior to
secretion(3, 4) . These variant chains are produced in
different cell types(5, 6) ; for example, 1,
2,
3,
4,
1,
2,
3,
1, and
2
chains are produced, but only the following combinations of chains have
thus far been observed:
1-
1-
1 (laminin),
2-
1-
1 (merosin),
1-
2-
1 (s-laminin),
2-
2-
1 (s-merosin),
3-
3-
2
(kalinin/nicein), and
4-
1-
1 (k-laminin).
Two models
of laminin-1 chain assembly, possibly relevant to the steps in
laminin-5 assembly, have been proposed. Peters et al.(3) and Morita et al.(4, 5) observed
a disulfide-linked 1
1 heterodimer as a presumed intermediate
and therefore suggested that the
1 chain was added at a later
stage. Alternatively, Wu et al.(7) reported that
initially laminin chains are assembled randomly.
Defects in
laminin-5 result in defective anchoring filaments, causing blistering
of the skin, and are now known to cause Herlitz junctional
epidermolysis bullosa (HJEB), ()an autosomal recessive
disorder characterized by generalized blister formation at the level of
the lamina lucida within the epidermal basement membrane (8, 9) . Recently, mutations in the
2 chain (10, 11) and
3 chain (12) genes of
laminin-5 have been reported in HJEB patients. Since laminin-5 is a
heterotrimer, knowledge of the steps in the assembly of the complete
protein may be important in future attempts to correlate specific chain
mutations with laminin-5 dysfunction and ultimately with clinical
phenotype. As a first step in understanding the pathophysiology of
HJEB, we have characterized the subunit assembly of laminin-5.
Our assembly study was conducted in a squamous cell carcinoma line (SCC-25). This cell line produces laminin-5 that is indistinguishable from that produced by normal human keratinocytes(2) . We determined the steps in laminin-5 chain assembly using endogenous laminin-5 and checked our conclusions using exogenous chains expressed from full-length cDNAs. We conducted immunoprecipitation reactions with general and chain-specific antibodies and used two-dimensional gels to resolve subunit association.
In the cell fraction
under nonreducing conditions, five forms of immunoreactive laminin-5
were observed (Fig. 1a, laneL). The
slowest migrating band is a cellular form () with an
estimated molecular mass of 460 kDa. Based on molecular mass and
immunoprecipitation by antisubunit antibodies (Fig. 1a, lanes
and
), the other forms in
the cell fraction were apparently dimers (
) and monomers
(
,
, and
) of laminin-5 subunits. Consistent with this
conclusion,
chain-specific antisera precipitated the
trimer,
dimer, and
monomer, but not
the
chain (Fig. 1a, lane
), whereas
-specific antisera recognized the
monomer as well as the
dimer and
trimer, but not the
monomer. Under reducing conditions, the same
samples showed three bands corresponding to the 200-kDa
3 (
), 145-kDa
3 (
), and 155-kDa
2 (
) subunits (Fig. 1b, lanesL,
, and
).
Figure 1:
Immunoprecipitation of SCC-25 cell
lysate and culture medium. SCC-25 cells were incubated in methionine-
and cystine-deficient medium for 2 h and then radiolabeled for 2 h
(cell lysate) or 24 h (culture medium) with 100 µCi/ml
[S]methionine and
[
S]cystine. Samples were immunoprecipitated with
anti-laminin-5 antibody (laneL), anti-
3
antibody (lane
) or anti-
2 antibody (lane
) as described under ``Experimental
Procedures.'' Each sample was boiled in SDS sample buffer either
not containing (nonreducing) or containing 2-mercaptoethanol
(reducing). Laminin-5 subunits were separated on 5% acrylamide gels and
visualized by fluorography. Processed forms of
and
chains
are indicated by
` and
`. a, cell
lysate, nonreducing conditions. The
heterotrimer,
heterodimer, and
,
, and
monomers are
visible. b, cell lysate, reducing conditions. The
,
, and
monomers are visible. c, culture medium,
nonreducing conditions. The
`
440-kDa medium form and
the
`
` 400-kDa medium form are visible. d,
culture medium, reducing conditions. The
`,
,
, and
` monomers are visible. A 250-kDa fibronectin band (FN)
is also visible.
Using
nonreducing conditions to analyze the conditioned medium (Fig. 1c, lanesL, ,
and
), two secreted forms of the laminin-5 molecule, the
440-kDa (
`
) and 400-kDa (
`
`) forms, were
observed (
` and
` are the processed forms of the
and
chains, respectively, whereas the
chain is not processed).
Monomers or dimers are probably not secreted since no bands are seen
even after longer exposures of the autoradiographs corresponding to the
molecular masses of either monomers or dimers. Under reducing
conditions, these same samples containing both forms of the
heterotrimer resolved into four bands, corresponding to 165-kDa
processed
3 (
`), 145-kDa
3 (
),
155-kDa
2 (
), and 105-kDa processed
2 (
`) (Fig. 1d, lanesL,
, and
). All three antibodies also
precipitated an additional band of 250 kDa under reducing conditions (Fig. 1d, FN). The intensity of this band was
diminished by preincubation of the samples with gelatin-Sepharose,
previously found to bind fibronectin. The 250-kDa band was present in
greater amounts when the preincubation step was deleted (data not
shown).
The extent of association of the laminin-5 subunits with
each other was assessed in cell lysates and culture medium by
two-dimensional SDS-PAGE under nonreducing conditions in the first
dimension and under reducing conditions in the second dimension. In the
cell lysate, the 460-kDa complex (; Fig. 2a, right-most band in the toppanel) observed under nonreducing conditions dissociated
under reducing conditions into an equimolar mixture of
3,
3,
and
2 subunits (lower left panel, spots directly below
band). The second band (
) from the origin under nonreducing
conditions dissociated into two forms of 145 and 155 kDa under reducing
conditions, indicative of a disulfide-linked heterodimer of
3 and
2. Monomers of the three subunits run under reducing conditions
are shown as markers in Fig. 2a (lowerrightpanel). The signal intensity of the
monomers under nonreducing conditions was
<
<
.
A weaker signal for
3 was reproducibly observed as compared with
3 (
) or
2 (
) monomers, perhaps
indicating that the intracellular pool of uncombined
3 is smaller
than that of the other chains. We cannot rule out the possibility of
preferential precipitation of the
or
chain by our general
antisera against laminin-5.
Figure 2:
Two-dimensional SDS-PAGE analysis of
precipitated laminin-5. SCC-25 cells were radiolabeled for either 2 h
(cell lysate) or 24 h (culture medium) and immunoprecipitated with
anti-laminin-5 antiserum. The samples were subjected to two-dimensional
SDS-PAGE as described under ``Experimental Procedures.''
Electrophoresis in the first dimension was performed under nonreducing
conditions, and electrophoresis in the second dimension was performed
under reducing conditions by applying the cut gel, infused with
2-mercaptoethanol, on top of the polyacrylamide slab. The opencircles denote the gel origins. Duplicate nonreducing (toppanels) and reducing (lower right
panels) gels are shown along each dimension to allow alignment of
the component chains seen in the second dimension gel. The positions of
molecular mass standards are shown relative to each of the first
dimension gels. a, cell lysate analyzed for laminin-5
components; b, culture medium analyzed for laminin-5
components. Laminin-5 subunits visible in the first dimension that were
resolved into component chains in the second dimension are the
heterotrimer; the
heterodimer; the
,
, and
monomers; the
`
440-kDa medium form;
and the
`
` 400-kDa medium
form.
In the medium (Fig. 2b),
the two forms of extracellularly processed laminin-5, 440 kDa
(`
) and 400 kDa (
`
`), were observed under
nonreducing conditions. Under reducing conditions, the 440-kDa form
(right-most band in the toppanel) dissociated into a
mixture of processed 165-kDa
3 and 140-kDa
3 and unprocessed
155-kDa
2 subunits. The 400-kDa laminin-5 (the second band from
the origin under nonreducing conditions) also dissociated into
processed
3 and
3, but displayed a complete substitution of
the processed 105-kDa
2 chain for unprocessed
2. The
nonspecific 250-kDa fibronectin band was again present under reducing
conditions.
Figure 3:
Pulse-chase kinetic analysis of laminin-5
biosynthesis. SCC-25 cells were pulsed with
[S]methionine and
[
S]cystine for 10 min and then incubated in
complete growth medium as a chase. After 0, 0.5, 1.5, 3, 6, and 24 h of
chase in unlabeled complete medium, either cell lysates or medium
samples were removed and then processed for immunoprecipitation with
anti-laminin-5 antiserum. The precipitates of immunoreactive laminin-5
forms were fractionated by SDS-PAGE after preparation under either
nonreducing or reducing conditions. a, cell fraction,
nonreducing conditions; b, cell fraction, reducing conditions; c, medium fraction, nonreducing conditions; d, medium
fraction, reducing conditions. Laminin-5 subunits visible are the
heterotrimer; the
heterodimer; the
,
, and
monomers; the
`
440-kDa medium form;
and the
`
` 400-kDa medium form. A 250-kDa fibronectin
band (FN) is also visible.
In the medium, after a 1.5-h chase, a 440-kDa band
was observed under nonreducing conditions (Fig. 3c).
The smaller 400-kDa form of laminin-5 appeared after a 3-h chase, and
its intensity steadily increased up to the 24-h chase period. A band
corresponding to the 460-kDa cellular form was not detected clearly.
The same samples under reducing conditions contained three bands
corresponding to the subunits of the 440-kDa form when observed after a
3-h chase (Fig. 3d). The processed 2 subunit
belonging to the 400-kDa form appeared after a 6-h chase. Two
additional protein bands of approximately 180 and 200 kDa appeared
during the chase period (Fig. 3d). These bands also
appeared in the 3- and 6-h chase lanes and decreased by 24 h (Fig. 1d and Fig. 2b) (reducing
conditions). The two bands are most likely intermediates of the
processed
3 chain since a 200-kDa protein is immunoblotted by an
anti-laminin-5 antibody and is present in pulse-chase experiments of
keratinocyte-conditioned medium(4) . A small amount of
laminin-5 containing the partially processed
3 chain may be
unresolvable from the 440- and 400-kDa laminin-5 bands on nonreduced
SDS-PAGE (Fig. 2b). The 250-kDa fibronectin band was
also seen in the 3-h chase lane and increased in intensity after the
24-h chase.
Figure 4:
Expression of recombinant 3 and
2 subunits in 293 cells. 293 cells were not transfected (lanes1, 7, and 13), transfected with the
pCPS vector alone (lanes 2, 8, and 14), or
cotransfected with pCPS and pCPS
3 (lanes3, 9, and 15), pCPS and pCPS
2 (lanes4, 10, and 16), or pCPS
3 and
pCPS
2 (lanes5, 11, and 17).
Two days later, the transfected cells were labeled with
[
S]methionine and
[
S]cystine for 2 h as described under
``Experimental Procedures.'' Labeled SCC-25 cells were
included as a marker for the positions of the laminin-5 chains (lanes6, 12, and 18). Cell
extracts were immunoprecipitated with anti-
3 antiserum (lanes
1-6), anti-
2 antiserum (lanes 7-12), or
anti-laminin-5 antiserum (Anti lam5; lanes
13-18) and resolved by SDS-PAGE after denaturation in SDS
sample buffer either under reducing (a) or nonreducing (b) conditions. (-), sample from untransfected
293 cells; V,
, and
, pCPS,
pCPS
3, and pCPS
2 used for transfection,
respectively.
The recombinant 3 chain was immunoprecipitated
from
3 cDNA-transfected 293 cells using anti-
3 antibody (Fig. 4a, lane3), but not from
untransfected cells or from cells transfected with the vector alone (Fig. 4a, lanes 1 and 2). As
expected, anti-
3 antibody did not recognize r
in
2
cDNA-transfected 293 cells (Fig. 4a, lane4); however, under reducing conditions,
immunoprecipitates from
3- and
2-cotransfected 293 cells
using the same antibody showed both r
and r
(Fig. 4a, lane5), providing evidence
for the formation of a
heterodimer. The recombinant chains
showed the same migration on SDS-PAGE under reducing conditions as
endogenous
3 and
2 from SCC-25 cells (Fig. 4a, lane6). Similar results
were obtained when the same set of transfection experiments were
immunoprecipitated with anti-
2 antibody. As expected,
2
antiserum did not react with r
, only with r
(Fig. 4a, lanes9 and 10),
and coprecipitated r
and r
from cotransfected 293 cells (lane11), providing further evidence of
heterodimer formation. Our anti-laminin-5 antibody was able to
recognize both individual recombinant chains (Fig. 4a, lanes15 and 16). In this case, recognition
of the two recombinant chains together in the cotransfection is not
necessarily the consequence of coprecipitation (Fig. 4a, lane17).
Under
nonreducing conditions, similar results were obtained, although
multimers of r and r
were evident in the cell extracts from
3- or
2-transfected 293 cells (Fig. 4b, lanes3, 5, 10, 11, 16, and 17). These high molecular mass bands
dissociated into bands of the same size as r
or r
monomers
upon electrophoresis under reducing conditions (Fig. 4a). Significantly, a heterodimer of r
and
r
was detected that could be distinguished from the multimer bands
in the extracts from
3- and
2-cotransfected 293 cells
(compare lanes3 and 5 and lanes10 and 11).
To further clarify the identity
of the presumed heterodimer, we performed two-dimensional
SDS-PAGE analysis with the samples from cotransfected cells. The
heterodimers of r
and r
have a similar molecular mass (300
kDa) to the homodimers of either r
(290 kDa) or r
(310 kDa).
We resolved the presumed heterodimer on a second dimension under
reducing conditions into its component r
and r
chains.
Anti-
3 antibody reacted with the r
monomer (Fig. 5a, blackasterisk) and
heterodimer (black and whitearrows). The
high molecular mass multimers dissociated into r
(Fig. 5a, blackarrowheads) and
r
(whitearrowheads).
Figure 5:
Interaction of recombinant 3 and
2 subunits in 293 cells. 293 cells cotransfected with recombinant
3 and
2 vectors were radiolabeled as described previously and
immunoprecipitated with either
3 or
2 chain-specific
antiserum. These samples were resolved by two-dimensional SDS-PAGE as
described under ``Experimental Procedures.'' Electrophoresis
in the first dimension was performed without reduction of disulfide
bonds. Electrophoresis in the second dimension was performed by
applying the cut gel, infused with 2-mercaptoethanol, on top of the
polyacrylamide slab. Duplicate nonreducing and reducing gels are shown
along each dimension. a, the same sample as in Fig. 4b (lane5) using anti-
3
antiserum; b, the same sample as in Fig. 4b (lane11) using anti-
2 antiserum. The blackasterisk shows the r
monomer. The black arrow shows r
resolved from the heterodimer. Blackarrowheads show r
resolved from
chain multimers. Whiteasterisks show the r
monomer. The whitearrow shows r
resolved from
the heterodimer. Whitearrowheads show r
resolved from
chain multimers. r
, recombinant
heterodimer containing both r
and
r
.
Anti-2 antibody
recognized both the r
monomer, which resolved into two bands (Fig. 5b, whiteasterisks), and
heterodimer (black and whitearrows). The
r
monomer resolved into two bands in the first dimension under
nonreducing conditions, probably due to the presence of an
intramolecular disulfide bond in the r
monomer. In the second
dimension (reducing conditions), the high molecular mass multimers
dissociated into only r
(Fig. 5b, white
arrowheads), with no r
-containing species visible. Although
the two antibodies showed different reactivity to the multimers,
cotransfected 293 cells definitely contain monomers (r
and
r
), multimers, and the heterodimer (r
r
).
In the presence of tunicamycin, monomers (p,
p, and
p), dimers (
p
p), and heterotrimers
(
p
p
p) migrated as lower molecular mass species as
compared with the untreated normal cell lysate samples under
nonreducing conditions (Fig. 6a, compare lanesN and T in the toppanel).
These presumably correspond to unglycosylated precursors as a result of
tunicamycin treatment. A similar observation was made under reducing
conditions, where each individual chain migrated as a lower molecular
mass species as compared with the untreated normal sample (Fig. 6a, compare lanesN and T in the lower right panel). The assembly of unglycosylated
laminin-5 subunits was assessed by two-dimensional SDS-PAGE. The
heterotrimer (
p
p
p), under nonreducing conditions,
dissociated upon reduction into a mixture of the unglycosylated
subunits (
p,
p, and
p), as did the heterodimer
(
p
p). These results suggest that in the absence of
glycosylation, the assembly of laminin-5 subunits occurs in the same
way as in untreated normal cells.
Figure 6:
Assembly and glycosylation of laminin-5
subunits. SCC-25 cells were radiolabeled as described previously in
medium containing either no addition or tunicamycin (3 mg/ml). The cell
lysate or culture medium was immunoprecipitated with anti-laminin-5
antiserum. Each sample was boiled in SDS sample buffer, electrophoresed
on 5% acrylamide, and visualized by fluorography. a, cell
lysate. Toppanel, nonreducing conditions, cells
either not treated (laneN) or tunicamycin-treated (laneT); lower right panel, reducing
conditions, cells either not treated or tunicamycin-treated; lower
left panel, two-dimensional SDS-PAGE analysis of laminin-5
immunoprecipitated from the tunicamycin-treated cell fraction.
Electrophoresis of same sample as in Fig. 4a (laneT) in the first dimension was performed without reduction
of disulfide bonds; electrophoresis in the second dimension was
performed by applying the cut gel, infused with 2-mercaptoethanol, on
top of the polyacrylamide slab. b, culture medium. Toppanel, nonreducing conditions, cells either not treated (laneN) or tunicamycin-treated (laneT); lower right panel, reducing conditions,
cells either not treated or tunicamycin-treated; lower left
panel, two-dimensional SDS-PAGE analysis of laminin-5
immunoprecipitated from the tunicamycin-treated medium fraction.
Electrophoresis of same sample as in Fig. 4d (laneT) in the first dimension was performed without reduction
of disulfide bonds; electrophoresis in the second dimension was
performed by applying the cut gel, infused with 2-mercaptoethanol, on
top of the polyacrylamide slab. Shown are the p
p
p
unglycosylated cellular form of laminin-5; the
p
p
unglycosylated heterodimer; the
p,
p, and
p
unglycosylated monomer precursors; the
p`
p
p
unglycosylated larger medium form of laminin-5; and the
p`
p
p` unglycosylated smaller medium form of
laminin-5.
Finally, we assessed the role of
glycosylation in the secretion and subsequent processing of laminin-5
by conducting a similar analysis as described above using the medium of
tunicamycin-treated cells. In SDS-PAGE of the medium from the
tunicamycin-treated cells under nonreducing conditions (Fig. 6b, laneT in the top
panel), two bands migrating as lower molecular mass species as
compared with the untreated normal medium fractions were observed,
corresponding to heterotrimers (p`
p
p and
p`
p
p`). Even after overexposure of this gel, no dimer
and monomer bands were seen. Under reducing conditions, the same
samples showed four fast migrating bands of unglycosylated proteins (Fig. 6b,
p`,
p,
p, and
p` in the lower right
panel). On two-dimensional SDS-PAGE analysis, the larger band of
first dimension electrophoresis dissociated into three proteins,
processed
3 (
p`), unprocessed
3 (
p), and unprocessed
2 (
p) precursors.
The lower signal dissociated into processed
3 (
p`),
unprocessed
3 (
p), processed
2 (
p`) precursors. Both the tunicamycin-treated cell and
medium fractions showed a small band estimated to be 80 kDa under
reducing conditions (Fig. 6, a and b, laneT in the lowerrightpanels).
Since both anti-
3 and anti-
2 reacted with this band on
immunoprecipitation (data not shown), it may be a product of
degradation of the heterotrimer, due to an increased instability of
unglycosylated precursors as compared with the normally glycosylated
chains.
We have characterized the subunit assembly of a novel
tissue-specific laminin variant, laminin-5, formerly known as nicein or
kalinin(1, 2) . Laminin-5 is present at
epithelial-stromal interfaces and is the primary component of the
anchoring filaments associated with hemidesmosomes and therefore is
crucial to the attachment of basal epithelial cells to the basement
membrane zone. Defects in laminin-5 have been shown to be a cause of a
lethal skin disease, Herlitz junctional epidermolysis
bullosa(10, 11, 12) . We utilized the
squamous cell carcinoma line SCC-25 as a source of constitutively high
levels of laminin-5 rather than cultured primary keratinocytes because
of the tendency of the keratinocytes to differentiate, resulting in a
down-regulation of laminin-5. As is the case for a variety of
multimeric proteins, we observed a specific order of addition for the
assembly of individual chains into heterotrimeric laminin-5. In the
cell fraction containing laminin-5 prior to secretion, we observed
monomers of each of the three subunits (3,
3, and
2), a
3
2 heterodimer, and an
3
3
2 heterotrimer. The
presence of a
heterodimer, but the absence of any other
heterodimeric species, was suggestive of an ordered assembly of
laminin-5 proceeding from the
heterodimer to the
heterotrimer by the addition of the
3 chain.
We employed a
eukaryotic expression vector to further test the possibility of a
stable association of the and
chains into a heterodimer as
an intermediate step in the assembly of the laminin-5 heterotrimer.
Earlier use of recombinant proteins to study assembly of other laminin
isotypes has been restricted to the use of the E8 fragment of mouse
Engelbreth-Holm-Swarm laminin(19) , prokaryotic expressed
Engelbreth-Holm-Swarm laminin(20) , and synthetic peptides (21) in vitro. Expression of recombinant proteins has
been an important tool in the study of subunit assembly of a variety of
proteins, however, including the interaction of fibrinogen subunits (22, 23) . Here we utilized complete cDNA expression
vectors for the
and
chains to establish that the two chains
stably heterodimerize in the absence of the
3 chain. Our
experiment also rules out the possibility that the
heterodimer is a breakdown product of the heterotrimer since assembly
occurs in the absence of the
3 chain. We used 293 cells for our
transfection experiments because they contain no endogenous laminin-5
chains and are transfectable at high efficiencies and yield high levels
of protein expression when exogenously added genes are driven by the
cytomegalovirus promoter. The fact that the human embryonic kidney cell
line 293 will express the
and
chains and allow their
interaction makes the involvement of any tissue-specific factors in
heterodimer formation unlikely.
The 3 monomer pool
was observed to be the smallest of the monomers (Fig. 1a (laneL), 2a, and 6 (a-c)). Pulse-chase experiments revealed that the
3
chain signal seemed to decay faster than the other two subunits (Fig. 3b). We postulate that the
3 chain is
limiting for heterotrimer assembly and that the association of the
3 chain with
3
2 heterodimers occurs rapidly to yield
complete heterotrimers. A precedent in laminin-1 from murine
teratocarcinoma cell lines has been shown in which the
1 chain is
limiting for assembly at the protein level, in accordance with its low
mRNA levels(24, 25) . One possible rationale for the
synthesis of the
3 chain being limiting in the synthesis of
laminin-5 is that the cell uses
chain synthesis as a determining
step in controlling the type of laminin to be assembled. Thus,
expression levels of either
1 or
2 may determine whether
laminin-1 (
1
1
1) or merosin (
2
1
1) is to be
assembled or whether s-laminin (
1
2
1) or s-merosin
(
2
2
1) is assembled. Although no laminin variant
containing
3
2 other than laminin-5 (
3
3
2) has
yet been described, the possibility of additional laminin variants
seems likely. One circumstance in which laminin-5 production would be
crucial is in activated keratinocytes repopulating a wound bed.
Keratinocytes in tissue culture display markers for the activated
phenotype as well, and the
3 chain pool typically exceeds the
other monomer pools in these cells (data not shown).
Following
assembly of the heterotrimer, processing of two out of the three chains
occurs, 3 and
2. The cytoplasmic form of laminin-5 was
previously identified as a 460-kDa precursor that contains unprocessed
forms of each chain(2) . Processing occurs after secretion,
with two predominant forms of 440 and 400 kDa present in the medium
fraction. Processing of the
3 chain to 165 kDa has already
occurred in both of these forms. However, in our pulse-chase
experiments, faint bands of 200 and 180 kDa were also visible (Fig. 3d, 3-, 6-, and 24-h chase samples). The 180-kDa
species may be an intermediate in the processing of the 200-kDa
3
chain to its final processed form of 165 kDa. Since these bands were
minor components, they presumably belong to a minor fraction of
heterotrimers secreted into the medium that were not yet processed at
the moment of sampling. Resolution of a small amount of the unprocessed
heterotrimer, and the presumed processing intermediate, may not be
readily resolvable from the major form of 440 kDa in these gels.
We
resolved the 440- and 400-kDa forms of heterotrimeric laminin-5 in the
medium fraction into the component chains in our SCC-25 cell model
system, as was previously done in keratinocytes (2) (Fig. 1c, lanesL, , and
). The larger species consists of
165-kDa
3, 145-kDa
3, and 155-kDa
2 chains, while the
smaller one consists of 165-kDa
3, 145-kDa
3, and 105-kDa
2 chains (Fig. 2b). Furthermore, in pulse-chase
experiments of the nonreduced medium samples (Fig. 3c),
the 440-kDa form of laminin-5 first appeared after the 1.5-h chase and
was clearly evident after the 3.0-h chase, whereas the 400-kDa form
appeared later, only after a 3-h chase, clearly visible after a 6-h
chase. Consistent with the 440- and 400-kDa forms being composed of the
same chains but with processed
2 (105 kDa) substituted for
unprocessed
2, the processed form of
2 (105 kDa) appears with
the same kinetics as the 400-kDa form. These results support a
precursor-product relationship between the 440- and 400-kDa forms, due
to processing of the
2 chain from 155 to 105 kDa. Dimers or
monomers were not detected in SCC-25 medium, consistent with a lack of
secretion in the absence of heterotrimer assembly, although these forms
might be present at steady-state concentrations below the detection
limit of the assay. A 250-kDa protein was coprecipitated with
laminin-5. Since the intensity of this signal was reduced by
preincubation with gelatin, this protein may be fibronectin.
Coprecipitation of fibronectin with laminin-5 was previously reported
in normal and HJEB human keratinocyte systems(26) .
Multimers of individual chains, such as a 1 chain multimer as
reported for mouse laminin-1(7) , were not detected in
laminin-5 from SCC-25 cells (Fig. 1a (lanesL,
, and
) and 2a).
It is possible that these forms might be present at steady-state
concentrations below the detection limits of the assay or are
immunologically unreactive. The latter possibility is less likely since
and
multimers were detected in transfected 293 cells by the
same antibodies. Multimer bands have previously been noted when other
proteins have been expressed in heterologous systems, for example the
expression of human fibrinogens
,
, and
in baby hamster
kidney cells(22) . In a study on the influenza hemagglutinin
protein, only properly folded multimeric proteins were transported out
of the rough endoplasmic reticulum, whereas incompletely folded
proteins either accumulated or were degraded in the endoplasmic
reticulum(27) . One characteristic of the 293 cells used to
conduct our transfection experiments may be a reduced capacity to deal
with improperly folded proteins.
We have shown that the assembly of
recombinant 3 and
2 chains is achieved without the presence
of the
3 chain (Fig. 4a, lanes 5 and 11). In a further analysis of the cotransfection products on
two-dimensional SDS gels, cells cotransfected with
3 and
2
expression vectors produced both monomers (Fig. 5, a and b, black and whiteasterisks) and the
heterodimer (Fig. 5, a and b, black and whitearrows). When anti-
3 antibody was used, the major
bands were
monomers (145 kDa) and
dimers (300 kDa).
The coexpression of
and
leads mainly to the formation of a
dimer held together by disulfide bond(s) as well as free
chain. When anti-
2 antibody was used, a
dimer
signal was also detected. Since this antibody does not recognize the
heteromultimer (Fig. 5a, black and whitearrows), heteromultimerization might obstruct
binding to antibody recognition sites. We conclude that the
3
chain is not necessary for
heterodimer formation, but that
it is an intermediate in heterotrimer formation in SCC-25 cells.
Considering that laminin-1 assembly probably proceeds via a
dimer, and from our results with laminin-5, it seems a reasonable
prediction that other laminin isoforms also will be shown to assemble
via a
dimer intermediate.
The pulse-chase study
demonstrated that newly synthesized laminin-5 subunits appear in the
cells immediately following a 10-min biosynthetic pulse with
[S]methionine and
[
S]cystine. Since other related proteins are
known to undergo addition of N-linked glycochains after a 1-h
chase, as reported for laminin-1 of human choriocarcinoma cells (3) and mouse embryonic carcinoma F9 cells(4) , we
expected to observe an increased mobility of the laminin-5 chains on
SDS-PAGE due to the lack of glycosylation of these intracellular
precursors. However, precursors with altered electrophoretic mobility
were not detected on two-dimensional SDS-PAGE (Fig. 2a)
and in the pulse-chase study (Fig. 3b). These results
suggest that the high mannose chains on the subunits are not processed
into complex forms.
To further investigate the role of glycosylation
in laminin-5 chain assembly, SCC-25 cells were treated with tunicamycin
to inhibit the addition of asparagine-linked carbohydrates, and cell
and medium fractions were analyzed by SDS-PAGE and two-dimensional
SDS-PAGE. The heterotrimer, heterodimer, and monomers were present in
the cell fraction, but had slightly increased mobility on the gels due
to the inhibition of glycosylation. The presence of the heterotrimer
suggested that the assembly of unglycosylated subunits also proceeds
through a 3
2 heterodimer to the
3
3
2
heterotrimer. Since protein disulfide-isomerase is present at the
luminal side of the rough endoplasmic reticulum(28) , disulfide
bond formation between laminin-5 subunits is expected to be completed
before they leave the rough endoplasmic reticulum. Additionally, it is
well established for N-glycosylated proteins that transfer of
high mannose-type oligosaccharide side chains en bloc proceeds
cotranslationally(29) . Dimer and trimer formation in the
presence of tunicamycin demonstrates that the inhibition of this
oligosaccharide transfer by tunicamycin does not affect subsequent
disulfide bond formation. However, high mannose oligosaccharide chains
can have a profound effect on the stability of
proteins(30, 31) , as has also been shown for
laminin-1(3) . The amount of laminin-5 in the medium fraction
was extremely small, so we postulate that N-glycosylation
protects laminin-5 polypeptides from nonspecific proteolytic
degradation.
Fig. 7presents a summary diagram of our
conclusions concerning post-transcriptional assembly and glycosylation
of laminin-5 subunits. These include the notions that 1) assembly
proceeds through a 3
2 heterodimer to the
3
3
2
heterotrimer; 2) synthesis of
3 polypeptides is the rate-limiting
step for assembly; 3) assembly of the subunits into a heterotrimer is
required for secretion; and 4) N-linked oligosaccharide chains
are not necessary for subunit assembly.
Figure 7:
Model of post-translational assembly and
glycosylation of laminin-5 subunits. Laminin-5 chain mRNAs are
translated into individual chain unglycosylated (thinlines) monomers, which are then glycosylated (thicklines). Heterodimers () assemble before the
addition of the
chain. Only heterotrimers are secreted regardless
of whether N-linked glycosylation is blocked by tunicamycin.
Proteolytic processing of the
and
chain occurs after
secretion. Laminin-5 intermediates observed in our experiments are as
follows: the
p
p
p unglycosylated cellular form of
laminin-5; the
p
p unglycosylated heterodimer; the
p,
p, and
p unglycosylated monomer precursors; the
p`
p
p unglycosylated larger medium form of laminin-5; and
the
p`
p
p` unglycosylated smaller medium form of
laminin-5. (CHO)
, core oligosaccharide side chain
transferred en bloc.
As reported(24) ,
HJEB patients have impaired expression of laminin-5, which is most
often a consequence of the defective synthesis of one of its subunits.
The disease is genetically heterogeneous, even considering the HJEB
patient population with laminin-5 defects, since any one or combination
of chain defects is possible within the heterotrimer. There is a range
of possible mutations in laminin-5 that may have effects on assembly,
stability, or secretion of the protein, and consequently, individual
patients might be expected to display different clinical phenotypes.
Chain assembly of laminins is mediated through the formation of
triple-stranded -helical coiled-coils, known to be among the
longest coiled-coil domains. In fibrinogen, another triple-stranded
-fibrous protein, the chain specificity is determined by
interactions between residues adjacent to the hydrophobic interaction
edges. Protein sequence data have been used to calculate ionic
interaction scores between heptad repeat regions in laminin-1 chains
that were in good agreement with experimental observations and that may
allow predictions of the stability of distinct laminin
isoforms(32, 33) . Now that we have noted the
appearance of a
3
2 heterodimer as a likely assembly
intermediate for laminin-5, we would expect some of the mutations
contained in HJEB patients to effect
3
2 dimerization, while
another class of mutations might interfere with
3 association with
the heterodimer to form a functional heterotrimer. We are currently
correlating HJEB patient phenotypes with characterization of molecular
defects to ascertain the extent to which we are able to use this
knowledge to predict clinical features and ultimately the course of the
disease.