(Received for publication, May 31, 1995; and in revised form, August 14, 1995)
From the
Small intestinal lactase-phlorizin hydrolase (LPH) is
synthesized as a large precursor (prepro-LPH) of 1926 amino acids. In
the endoplasmic reticulum, prepro-LPH is split by signal protease. The
resulting pro-LPH is cut to mature LPH directly (human) or via a
180-kDa intermediate (rabbit), most likely in the trans-Golgi
network or in a later compartment. Antibodies directed against
different regions of rabbit pro-LPH locate the cleavage site
resulting in the 180-kDa intermediate between amino acid residues 79
and 286. This stretch contains the two sequences
-Arg-Cys-Tyr-Arg and
-Arg-Ala-Ser-Arg
, which are potential cleavage sites
for subtilisin-like proprotein convertases. These sites are not
conserved in human pro-LPH. By coexpression in COS 7 cells of rabbit
prepro-LPH and proprotein convertases (PC1/3, PC2, PC6A, PC6B, furin),
we show that furin, PC1/3, and PC6A generate a processing intermediate
that is immunologically indistinguishable from the one observed in
vivo. Furin, PC1/3, and PC6A are all expressed in the small
intestine as shown by a polymerase chain reaction-based approach and,
more specifically, in enterocytes, as shown by in situ hybridization. These results suggest that furin, PC1/3, and/or
PC6A are responsible for the in vivo processing of rabbit
pro-LPH to the 180-kDa intermediate.
Small intestinal lactase-phlorizin hydrolase (LPH, ()EC 3.2.1.23-26) with an apparent molecular mass of
135 kDa and 160 kDa in rabbit and human, respectively, is synthesized
as a very large precursor of 1926 (rabbit) or 1927 (human) amino acid
residues(1) . This precursor (prepro-LPH) begins with a typical
signal sequence which is split at position 19-20 (1, 2) ; it ends with a membrane-spanning domain (1, 3) and a cytosolic C-terminal
sequence(4) . Between the signal sequence and the membrane
anchor, four homologous regions (I to IV) can be
recognized(1) . Only region III (lactase), region IV (phlorizin
hydrolase)(3) , the membrane-spanning segment, and the
cytosolic C terminus make the ``mature'' LPH which can be
isolated from the brush-border membrane. Processing of pro-LPH to LPH
takes place intracellularly(5, 6, 7) (although pancreatic proteases also may contribute to
it(8, 9) ), in one step in human (5, 7, 10) or two steps in the
rabbit(11, 12) . In the latter species, an
intermediate is formed (apparent molecular mass 180 kDa), which is
split to yield mature LPH of 135 kDa, beginning at position 867 of
prepro-LPH(1) . In human, pro-LPH gives rise in one step to
mature LPH, the final secondary N terminus being at position
868(13) .
In the rabbit and human, the amino acid
sequence immediately upstream of the N terminus of mature LPH is SKTR
and SKVR, respectively(1) . We will not deal here with the
processing at this position.
In the present paper we have investigated the first proteolytic processing step of rabbit pro-LPH, i.e. the conversion of pro-LPH to the intermediate 180-kDa form. The converting protease(s) responsible for this step have been identified as furin, PC1/3, and/or PC6A. The presence of their transcripts in the rabbit small intestine was confirmed by a polymerase chain reaction approach and by Northern analysis. By in situ hybridization, we show that all three proprotein convertases are expressed in enterocytes, the very cells in which LPH is expressed and processed.
pSCTmLPH (human prepro-LPH(14) ) and pRB-1R (rabbit prepro-LPH(15) ) were prepared in our laboratory. The cDNAs coding for different proprotein convertases (PCs) were kindly provided by the following people: pSCTmfurin (mouse furin (16) (subcloned into the vector pSCT Gal-X-556(17) )), pCMVPC6A (mouse PC6A(18) ), and pCMVPC6B (mouse PC6B (19) ) were provided by Dr. K. Nakayama, University of Tsukuba, Japan. pCD-RPC3 (rat PC1/3(20) ) and pCD-RPC2 (rat PC2(20) ) were from Dr. N. P. Birch, University of Auckland, New Zealand. pRcCMV-PC1/3 (mouse PC1/3(21) ) and pRcCMV-PC2 (mouse PC2(22) ) were obtained from Dr. N. G. Seidah, Clinical Research Institute of Montreal, Canada.
For immunoverlays (see below), the cells were
collected 48 h after transfection by detachment with a rubber
policeman. Cells were solubilized in 5 Laemmli sample buffer,
boiled for 2 min, and then used for SDS-PAGE analysis.
For metabolic
labeling studies, the cells were cultured for another 24 h in fresh
medium. 72 h after transfection, the cells were washed with MEM, and
then incubated in 2 ml of labeling medium (MEM supplemented with 10%
dialyzed FCS) for 60 min at 37 °C and 5% CO. After
depletion of methionine, the cells were pulse-labeled in 1 ml of
labeling medium, containing 25 µCi of
[
S]methionine (Amersham) for 1 h. The cells were
washed twice with PBS and were then either directly collected or chased
in DMEM/10% FCS supplemented with 10 mM cold methionine. (In
some experiments, BFA was added to all media at a concentration of 5
µg/ml.) The labeled cells were collected and solubilized in 400
µl of lysis buffer (25 mM Tris, pH 8.0, 50 mM NaCl, 1% deoxycholate, 1% Nonidet P-40, 0.01 volume of 100
mM phenylmethylsulfonyl fluoride, 0.01 volume of inhibitor
mixture (0.25 mg/ml pepstatin, 0.06 mg/ml aprotinin, 1.1 mg/ml
leupeptin, 4.7 mg/ml benzamidine, 0.24 mg/ml bestatin, 0.3 mg/ml E-64,
and 38.3 mg/ml o-phenanthroline)). Immunoprecipitations, using
polyclonal guinea pig-anti rabbit LPH (see above) or monoclonal
mouse-anti human LPH (27) antibodies, were performed as
described by Lottaz et al.(23) .
Degenerate primers were designed based on the conserved amino acid sequences within the catalytic domain of known subtilisin-like endoproteases(29) . Two upstream primers, SGrlac3+ (5`-CAYGGIACNMGNTGYGC, corresponding to the His-Gly-Thr-Arg-Cys-Ala motif, where M = (A/C), R = (A/G), W = (A/T), Y = (C/T), K = (G/T), S = (G/C), N = (A/G/C/T)), and SGrlac6+ (5`-TAYWSIGCIWSNTGGGG, corresponding to the Tyr-Ser-Ala-Ser-Trp-Gly motif) and two downstream primers, SGrlac4- (5`-TGCATRTCNCKCCANGT, corresponding to the Thr-Trp-Arg-Asp-Met-Gln motif) and SGrlac5- (5`-TGNACRTCNCKCCANGT, corresponding to the Thr-Trp-Arg-Asp-Val-Gln motif) were used in PCR for amplification using Taq DNA polymerase (Perkin Elmer).
In the first reaction
(67 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate,
6.7 mM MgCl, 10 mM 2-mercaptoethanol, 1
mM dNTP, 10% dimethyl sulfoxide, 0.72% bovine serum albumin,
and 1.2 µM concentration of each primer), primers
SGrlac3+ and SGrlac4- were used and samples were taken
through 35 cycles of 1 min at 94 °C, 90 s at 40 °C, and 90 s at
65 °C. This reaction produced a 600-base pair DNA fragment. In the
second reaction, primers SGrlac6+ and SGrlac4- or
SGrlac5- were used. 1 µl of a 1/10 dilution of the first
reaction was taken through 35 cycles of 1 min at 94 °C, 90 s at 40
°C, and 90 s at 65 °C. This second reaction produced two bands
of 400 and 600 base pairs, respectively, which were cloned into
pBluescript by blunt end ligation.
Clones were sequenced using T7 or T3 primers and the DNA sequencing kit from U. S. Biochemical Corp. Nucleotide sequences were compared with the Gene/EMBL Data Bank using the GCG software package (University of Wisconsin).
RNA probes were
synthesized in the presence of biotin-11-UTP (Boehringer) from the
rabbit cDNA clones described above. The sizes of these cRNAs were
approximately 600, 400, 600, and 600 nucleotides for furin, PC1/3, PC2,
and PC6A/B, respectively. Their sizes were reduced to about 150
nucleotides by alkaline hydrolysis. Hybridization was performed in 50%
formamide, 5 SSC, 5
Denhardt's solution, 10%
dextran sulfate, 200 µg/ml salmon sperm DNA, pH 6.1. The in
situ hybridization experiments were performed in a fully automated
way, using the VENTANA ISH System (Ventana Medical Systems, Tucson,
AZ). After a proteinase pretreatment step with protease 1 (Ventana
Medical Systems) for 8 min at 37 °C, the slides were washed and
dried. After a light denaturing step at 50 °C for 1 min, the
hybridization was performed for 60 min at 47 °C. The slides were
washed twice with 2
SSC, once with 0.5
SSC, twice with
0.1
SSC (all at 45 °C) and then incubated with 20 µg/ml
RNase A (Sigma) for 12 min at 37 °C. The slides were incubated with
streptavidin-peroxidase for 12 min at 42 °C. The peroxidase
substrate (Ventana Medical Systems), aminoethylcarbonate, and
H
O
were applied for 20 min at 42 °C. The
slides were removed from the machine and slightly counterstained with
Mayer's hemalum solution (Merck) prior to mounting with aquamount
solution (BDH).
Figure 1: SDS-PAGE analysis of LPH synthesized by explants of rabbit small intestine. A, pieces from the proximal small intestine of a 6-month-old male rabbit were metabolically labeled for 1 h and then chased for 1 to 20 h as indicated. The different LPH forms were immunoisolated using an antibody directed against mature LPH(3) . B, pieces of medial small intestine from a 3.5-year-old male rabbit were continuously labeled for 6 h. The different LPH forms were then isolated using antibodies directed against segments Ia, Ib, and II and against mature LPH (m). C, schematic drawing of rabbit prepro-LPH. Putative cleavage sites and peptides used to raise antibodies are indicated below and above the figure, respectively.
Most prohormones and neuroendocrine precursor proteins of
the regulated pathway are processed at KR or RR
sequences
(
indicating the cleavage site), while the precursors of some
growth factors and a few plasma membrane proteins (that are delivered
via the constitutive pathway) are processed at more complex multibasic
cleavage sites of the general type RX(K/R)R
. However,
cleavage after RXXR
has been reported in a number of
cases (see e.g.(37) and (38) ).
As to
pro-LPH, in the stretch between positions 78 and 287 (i.e. between the sequences of the peptides used to raise antibodies in
the experiment above, see Fig. 1C), rabbit pro-LPH
contains two sequences of the RXXR type, which are
potential cleavage sites for furin or other PCs:
-Arg-Ala-Ser-Arg
and
-Arg-Cys-Tyr-Arg
. Neither site is present in the
human enzyme, which is split to final LPH without going through this
intermediate. Human pro-LPH, however, does have two dibasic sites
between regions Ia and Ib that could in principle serve as a cleavage
signal (-Arg-Arg
and -Arg-Arg
).
COS cells transiently transfected with human prepro-LPH cDNA produce, but proteolytically process pro-LPH to mature LPH only to a very low extent(23, 39) . This cell line, therefore, is suitable to study the effect of PCs on the processing of pro-LPH. We thus coexpressed in COS 7 cells rabbit or human prepro-LPH along with individual PCs (furin, PC1/3, PC2, PC6A, PC6B; see Table 1) of rat and mouse origin.
The results of the coexpression experiments ( Fig. 2and Table 1) clearly show that coexpression of rabbit prepro-LPH with furin and, but to a lesser extent, PC1/3 or PC6A leads to the appearance of a band of approximately 180 kDa. Furin converts more than 55% of 220-kDa pro-LPH into a band with a size identical with the 180-kDa intermediate found in organ culture, whereas PC1/3 and PC6A convert about 38% and 18% of pro-LPH, respectively. All other proteases tested do not cleave the 220-kDa protein in significant amounts. Note that the processing of pro-LPH in general is slow, since in organ cultures even after a 20-h chase there is still some pro-LPH left (cf. Fig. 1A).
Figure 2:
SDS-PAGE analysis of immunoisolated rabbit
and human LPH coexpressed in COS 7 cells together with proprotein
convertases. COS 7 cells were transiently transfected with cDNAs coding
for rabbit (top) or human (bottom) prepro-LPH (70% of
total DNA) together with cDNAs coding for PCs (30% of total DNA) as
indicated. 72 h after transfection, the cells were pulse-labeled for 1
h in medium containing 25 µCi of
[S]methionine and then chased for 5 h in
complete medium supplemented with 10 mM methionine. Rabbit and
human LPH were immunoprecipitated from solubilized cells using
polyclonal guinea pig anti-rabbit LPH (3) and monoclonal
anti-human LPH antibodies(27) , respectively. Note that in the
chased furin sample, the low amount of rabbit LPH protein is not due to
degradation of LPH, but is due to a lower amount of COS 7 cells
used.
Interestingly, human pro-LPH (which is directly converted into mature LPH) is not cleaved by any of the PCs tested, providing a ``negative control'' that furin or PC1/3 or PC6A might indeed be the protease(s) generating the 180-kDa intermediate in rabbit LPH. When human prepro-LPH is cotransfected with PCs, a band of approximately 150 kDa is sometimes detectable (Fig. 2). This band, however, is also visible in cells transfected with human prepro-LPH alone, indicating that human pro-LPH might be processed to some extent (or degraded) by an intrinsic protease of the COS 7 cells.
Since processing of pro-LPH is known to take place in the trans-Golgi network (TGN) or in a later compartment(23, 40, 41) , we wondered whether this was also true in COS 7 cells overexpressing furin. While the intracellular location of endogenous furin has not been reported (due to its very low level of expression), recombinant furin, expressed after transfection, has been localized to the Golgi complex by immunofluorescence (42, 43) and found to be concentrated in the TGN by immunoelectron microscopy(44) . If this is true also for transfected COS 7 cells, then the processing of rabbit pro-LPH should be prevented by brefeldin A (BFA), since BFA interrupts the transport from the trans-Golgi to the TGN. The processing of rabbit pro-LPH to the 180-kDa intermediate was completely blocked in the presence of 5 µg/ml BFA (Fig. 3). The conclusions therefore are (i) that overexpressed furin is most likely localized in the TGN in COS 7 cells, and (ii) that the processing pattern in the presence or absence of BFA is indistinguishable from the one reported in organ cultures of rabbit small intestine(41) .
Figure 3:
BFA
blocks the processing by furin of pro-LPH to the 180-kDa form. COS 7
cells were transiently transfected with rabbit LPH-cDNA alone or
together with furin-cDNA (30% of total DNA). 72 h after transfection,
the cells were metabolically labeled for 60 min in medium containing 25
µCi of [S]methionine and then chased for 4 h
in complete medium containing 10 mM methionine. All
incubations were either in the presence (+) or absence(-) of
5 µg/ml BFA. Rabbit LPH was immunoprecipitated and then analyzed by
SDS-PAGE and autoradiography.
In analogy to the experiment shown in Fig. 1B, we tried to localize the cleavage site within region I of rabbit pro-LPH using the antibodies generated against regions Ia, Ib, and II. We cotransfected COS 7 cells with rabbit prepro-LPH and all available PCs and detected the different LPH forms on Western blots. The results for furin are shown in Fig. 4. In cells transfected with rabbit prepro-LPH alone, only the 220-kDa band is detectable with all antibodies (there is an additional faint band of approximately 200 kDa; probably high mannose glycosylated pro-LPH). Coexpression with furin leads to the appearance of an additional band at approximately 180 kDa. This band is not detected by the antibody against region Ia, weakly detected by the antibody against region Ib, and clearly detected by the antibody against region II. The same pattern was observed for PC1/3 (data not shown), the 180-kDa band being much weaker due to the lower extent of processing (see Table 1). No band at 180 kDa could be detected for PC2 and PC6B (not shown).
Figure 4:
Immunodetection of different rabbit LPH
forms in COS 7 cells transfected with furin. COS 7 cells were
transiently cotransfected with cDNAs coding for rabbit prepro-LPH (70%
of total DNA) and furin (30% of total DNA) as indicated. Cells were
collected 48 h after transfection and solubilized in 5 Laemmli
sample buffer. Proteins were separated by SDS-PAGE and then transferred
onto a polyvinylidene difluoride membrane. The different LPH forms were
detected using antibodies directed against regions Ia (left),
Ib (middle), and II (right), respectively. The
positions of pro-LPH and the 180-kDa intermediate are marked by an arrow and arrowhead,
respectively.
Thus, the pattern of LPH bands precipitated by our battery of antibodies is identical in cultures of rabbit small intestine (Fig. 1B) and in COS 7 cells coexpressing rabbit prepro-LPH and furin.
Since furin is the enzyme that cleaves rabbit pro-LPH most efficiently, we looked at its expression by Northern blotting (not shown). Rabbit furin is encoded by two mRNAs of approximately 5 and 6 kilobases, respectively, the former being the more abundant. These sizes are slightly larger than those found for furin in mouse and rat(46) .
Figure 5:
Detection of PC transcripts by in situ hybridization in rabbit tissues. Furin, PC1/3, PC2, and PC6A/B
transcripts were detected with biotinylated riboprobes and
streptavidin-peroxidase (brown reaction product). Nuclei were
counterstained with Mayer's hemalum solution (violet
color). All samples were, unless otherwise indicated, treated with
RNase A after the hybridization step to eliminate nonspecific labeling. A, furin in the duodenum (photographed at a 10
magnification); B, PC1/3 in the jejunum (
25); C, PC5/6 in the jejunum (
10); D, PC2 in the
ileum (
10); E, PC1/3 in the stomach (
10); F, furin in the pancreas (
10), hybridization with the
probe was preceded by an RNase A treatment; G, furin in the
stomach (
10), no RNase A treatment after the hybridization; H, LPH in the stomach (
10); I, PC1/3 in the
liver (
25); J, furin in the pancreas (
40), no
RNase A treatment after the hybridization; K, PC1/3 in the
stomach (
25); L, PC1/3 in the stomach (
100).
These experiments clearly show that furin, PC1/3, and PC6A transcripts are present in the enterocytes of the small intestine. Thus, one or more of these enzymes are most likely to process rabbit pro-LPH to the 180-kDa intermediate.
Small intestinal rabbit pro-LPH, contrary to human pro-LPH, is subjected to at least two proteolytic processing steps, rather than one, on its way from the endoplasmic reticulum to the brush-border membrane. In the rabbit, an intermediate of 180 kDa is formed. The objective of the present work has been that of identifying the protease(s) involved in the formation of this intermediate from pro-LPH.
Our conclusions are that rabbit enterocytes process pro-LPH
to the 180-kDa intermediate by way of furin and possibly PC1/3 and/or
PC6A for the following reasons. First (Fig. 1B), rabbit
intestinal pro-LPH is cut within a protein region where furin, PC1/3,
and PC6A consensus sequences occur, namely
-Arg-Cys-Tyr-Arg and
-Arg-Ala-Ser-Arg
. Of these sequences, the latter is
the more probable since cysteine residues have been found to be
excluded from position -3(47, 48) . Neither of
these two potential cleavage sequences occurs in human pro-LPH which is
processed to mature LPH directly. Second (Fig. 2), coexpression
of rabbit pro-LPH and these PCs in COS 7 cells leads to the appearance
of the 180-kDa intermediate; PC2 and PC6B do not. Third (Fig. 2), none of these proteases cleaves human pro-LPH in which
the consensus sequences indicated above do not occur. Fourth, by
reverse transcription-PCR, we show that furin, PC1/3, and PC6A/B
transcripts are present in the small intestine and, more specifically,
as shown by in situ hybridization (Fig. 5), in
enterocytes (the very cells which express and proteolytically process
pro-LPH). The distribution of these transcripts varies along the length
of the intestine (as does the distribution of pro-LPH transcripts), but
generally, stronger signals are detected for furin and PC1/3 than for
PC6A/B. Fifth (Fig. 3), we also show that the processing of
pro-LPH to the 180-kDa intermediate by way of furin occurs in the TGN,
or, thereafter, that is in the same or in a closely related compartment
where we have localized the proteolytic processing of human pro-LPH to
the mature enzyme(23) .
Walker et al.(49) have suggested that
furin may show a broader specificity when overexpressed. However, this
observation does not change our conclusions, since rabbit pro-LPH is
still efficiently processed even in the presence of a 5000-fold lower
amount of furin cDNA (not shown). In addition, experiments in
Ltk cells which endogenously express furin, but not
the other PCs(46) , show that overexpressed rabbit, but not
human pro-LPH, is processed to the 180-kDa intermediate in these
cells.
PC6A and PC6B contain the same catalytic domain. Interestingly, only PC6A, which is a soluble enzyme, cleaves rabbit pro-LPH, whereas PC6B, which contains a C-terminal membrane anchor(19) , does not. Since pro-LPH is anchored to the same membrane as PC6B, this may indicate a steric problem in the processing of pro-LPH by PC6B or absence of direct physical interaction. Furin, which also contains a membrane anchor, cleaves pro-LPH, which may indicate that the interaction between pro-LPH and furin does not suffer from a similar steric problem. Alternatively, the cleavage of pro-LPH may be due to the truncated, soluble form of the enzyme (see e.g.(50) ). Whatever form of furin splits pro-LPH, furin and pro-LPH must at some point colocalize.
In order to see whether furin
cleaves rabbit pro-LPH at the authentic site in COS 7 cells, we have
analyzed the different LPH forms obtained in organ cultures and in COS
7 cells on the same polyacrylamide gel. The migration behavior of
pro-LPH and the 180-kDa intermediate was indistinguishable between the
two samples, both before and after deglycosylation with N-glycosidase F (not shown). These findings further support
the idea that furin is involved in the generation of the 180-kDa
intermediate and that cleavage occurs at the
-Arg-Ala-Ser-Arg site.
Furin and the other
subtilisin-like PCs tested in the present work do not cleave this site
leading to mature LPH in rabbit. In some experiments, we have detected
a band of approximately 150 kDa, i.e. with a size similar to
mature LPH (cf. Fig. 2). This band, however, is also
present in COS 7 cells which are transfected with rabbit or human
prepro-LPH only, suggesting that this polypeptide is generated by an
endogenous protease of COS 7 cells (see also Refs. 23, 39, 53). Both
rabbit and human pro-LPH have a RXXR motif approximately
28 amino acids upstream of the mature cleavage site. This site might be
used by an endogenous protease as well as by furin (at least in the
case of human pro-LPH). We do not know why rabbit pro-LPH is not
further processed at this site by furin or any other PC used in the
experiments. Further sequence constraints, however, which are different
between the human and rabbit enzyme may play an important role. Indeed,
as shown recently, replacement of amino acids close to the cleavage
site in stromelysin-3 strongly influences the furin-dependent
processing of this protease(53) .
Pancreatic proteases play
a minor role, if at all, in the processing of pro-LPH ((9) and
references therein). Granzyme A, a potential candidate (54) ,
shows trypsin-like activity and, at least in vitro, cleaves
synthetic substrates with basic amino acids in positions -1 and
-3. ()However, by coexpression of rabbit or human
prepro-LPH together with a cDNA coding for mouse granzyme A in COS 7
cells, and also by an in vitro assay with pure pro-LPH and
pure granzyme A, we did not find any processing of pro-LPH.
Other candidates could comprise duodenase, a serine protease with
trypsin- and chymotrypsin-like activities that has been isolated
recently from bovine duodenal mucosa(55) . Further candidates
are homologues of the arginine-selective yeast aspartic
protease(56) , or an arginine-selective endoprotease that was
isolated from rat intestinal mucosa(57, 58) .
Involvement of enzymes from the latter groups would, however, imply
that final processing of rabbit pro-LPH occurs in the intestinal lumen
by a protease of the enterocytes.