In contrast to an 80 kDa mature form of the wild-type and TNSALPR433H, a unique disulfide-bonded 160 kDa molecular species appeared on the cell surface of the cells expressing TNSALPR433C
Trang 1alkaline phosphatase with an Arg433 fi Cys substitution associated with severe hypophosphatasia
Makiko Nasu1, Masahiro Ito2, Yoko Ishida2, Natsuko Numa3, Keiichi Komaru4, Shuichi Nomura1 and Kimimitsu Oda2,5
1 Division of Oral Health in Aging and Fixed Prosthodontics, Niigata University Graduate School of Medical and Dental Sciences, Japan
2 Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan
3 Division of Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, Japan
4 Kitasato Junior College of Health and Hygienic Sciences, Yamatomachi, Minami-Uonuma-shi, Niigata, Japan
5 Center for Transdisciplinary Research, Niigata University, Japan
Keywords
alkaline phosphatase; bone; disulfide bridge;
hypophosphatasia; loss of function
Correspondence
K Oda, Division of Oral Biochemistry,
Niigata University Graduate School of
Medical and Dental Sciences, 2-5274,
Gakkocho-dori Niigata 951-8514, Japan
Fax: +81 25 227 0803
Tel: +81 25 227 2827
E-mail: oda@dent.niigata-u.ac.jp
(Received 24 September 2006, accepted
23 October 2006)
doi:10.1111/j.1742-4658.2006.05550.x
Various mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia characterized by defective bone and tooth mineralization; however, the underlying molecular mechanisms remain largely to be elucidated Substitution of an arginine at position 433 with a histidine [TNSALP(R433H)] or a cysteine [TNSALP(R433C)] was reported in patients diagnosed with the mild or severe form of hypo-phosphatasia, respectively To define the molecular phenotype of the two TNSALP mutants, we sought to examine them in transient (COS-1) and conditional (CHO-K1 Tet-On) heterologous expression systems In contrast
to an 80 kDa mature form of the wild-type and TNSALP(R433H), a unique disulfide-bonded 160 kDa molecular species appeared on the cell surface
of the cells expressing TNSALP(R433C) Sucrose density gradient centri-fugation demonstrated that TNSALP(R433C) forms a disulfide-bonded dimer, instead of being noncovalently assembled like the wild-type Of the five cysteine residues per subunit of the wild-type, only Cys102 is thought to
be present in a free form Replacement of Cys102 with serine did not affect the dimerization state of TNSALP(R433C), implying that TNSALP(R433C) forms a disulfide bridge between the cysteine residues at position 433 on each subunit Although the cross-linking did not significantly interfere with the intracellular transport and cell surface expression of TNSALP(R433C),
it strongly inhibited its alkaline phosphatase activity This is in contrast to TNSALP(R433H), which shows enzyme activity comparable to that of the wild-type Importantly, addition of dithiothreitol to the culture medium was found to partially reduce the amount of the cross-linked form in the cells expressing TNSALP(R433C), concomitantly with a significant increase in enzyme activity, suggesting that the cross-link between two subunits distorts the overall structure of the enzyme such that it no longer efficiently carries out its catalytic function Increased susceptibility to proteases confirmed a
Abbreviations
Endo H, endo-b-N-acetylglucosaminidase H; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol, PI-PLC, phosphatidylinositol-specific phospholipase C; TNSALP, tissue-nonphosphatidylinositol-specific alkaline phosphatase; TNSALP(R433C), tissue-nonphosphatidylinositol-specific alkaline phosphatase with
an arginine to cysteine substitution at position 433; TNSALP(R433H), tissue-nonspecific alkaline phosphatase with an arginine to histidine substitution at position 433.
Trang 2Hypophosphatasia is characterized by defective
osteo-genesis with various degree of failure in mineralization
of hard tissues such as bone and tooth [1–3] Various
mutations in the human tissue-nonspecific alkaline
phosphatase (TNSALP, EC 3.1.3.1) gene are thought
to be responsible for hypophosphatasia [1–5]
Hypo-phosphatasia is customarily divided into: (a) perinatal
hypophosphatasia; (b) infantile hypophosphatasia; (c)
childhood hypophosphatasia; (d) adult
hypophospha-tasia; and (e) odonto-type hypophosphatasia The
bio-chemical hallmark of the disease is reduction in serum
alkaline phosphatase activity Variation in clinical
expression is known to correlate well with variable
residual enzymatic activities in hypophosphatasia
patients (6,7) In general, the lower the activity, the
more severe the symptoms As of 24 July 2006, 184
mutations had been reported in the TNSALP gene
worldwide, and about 80% of them are missense
muta-tions [7] (http://www.sesep.usvq.fr.⁄ Database.html)
Recently, using a computer-assisted, three-dimensional
model of TNSALP, Mornet et al have proposed the
categorization of missense mutations into different
functional domains, such as the active site, the
homodimer interface and the crown domain [8] It is
now easier to predict, estimate and probably
under-stand the effects of some of the missense mutations on
the TNSALP molecule However, the structural
evidence in itself may not be sufficient to assess the
effects of other mutations on TNSALP, especially if a
particular amino acid plays an essential role in the
adoption of the native structure other than its role in
maintaining the structure and function of the fully
folded enzyme In this respect, we previously reported
that several TNSALP mutant proteins, which were
reported in severe hypophosphatasia patients, tend to
form a high molecular mass aggregate in the
endoplas-mic reticulum (ER), resulting in decreased cell surface
appearance of the TNSALP mutants, suggesting
impairment of the folding and assembly process for
TNSALP [9–13] Furthermore, some mutant proteins
undergo proteasomal degradation [11–13] Obviously,
an ER exit defect could be an important factor in
the etiology of severe forms of hypophosphatasia,
irrespective of whether mutant enzymes exhibit
vari-able residual enzyme activity [3] TNSALP is an
ectoenzyme anchored to the plasma membrane via
glycosylphosphatidylinositol (GPI), and is believed to regulate biomineralization by hydrolyzing inorganic pyrophosphate, the extracellular matrix mineralization inhibitor, on the surface of osteoblasts, chondrocytes and matrix vesicles derived from them [3,14]
TNSALP(R433H arginine to histidine substitution) was found in a compound heterozygote (R433H⁄ D389G) diagnosed with odontohypophosphatasia [15], whereas TNSALP(R433C arginine to cysteine substitu-tion) was found in two independent homozygous patients with infantile hypophosphatasia [16] The three-dimensional structure of human TNSALP predicts that an arginine residue at position 433 is unique to TNSALP and is located at the entrance of the active site pocket, raising the possibility of its involvement in sub-strate positioning [8] Because of its conservative nature, the replacement of arginine with histidine was assumed
to affect the catalytic function of TNSALP less severely than replacement with cysteine Here, we report that both TNSALP(R433H) and TNSALP(R433C) are anchored to the plasma membrane via GPI, like the wild-type Nonetheless, in contrast to the wild-type and TNSALP(R433H), TNSALP(R433C) forms a
covalent-ly cross-linked dimer with low catacovalent-lytic efficiency, pre-sumably explaining the severity of the disease when this particular mutation is present in a homozygous state
Results
Transient expression of TNSALP mutants
in COS-1 cells Human TNSALP folds and assembles as a noncova-lently associated homodimer in the ER and then pro-ceeds through the secretory pathway to the plasma membrane, where it is anchored via GPI [9,10] Of five potential N-glycosylation sites of TNSALP, three sites are attached by oligosaccharide chains when the pro-tein is expressed in COS-1 cells [9] TNSALP is syn-thesized as a 66 kDa endo-b-N-glucosaminidase H (Endo H)-sensitive form, is processed to a mature
80 kDa Endo H-resistant form, and finally appears on the cell surface To examine whether the two missense mutations at position 433 of TNSALP affect the biosynthesis of TNSALP, we transfected COS-1 cells with a plasmid encoding TNSALP(R433C) or
gross conformational change of TNSALP(R433C) compared with the wild-type Thus, loss of function resulting from the interchain disulfide bridge is the molecular basis for the lethal hypophosphatasia associated with TNS-ALP(R433C)
Trang 3TNSALP(R433H) The cells were metabolically labeled
with [35S]methionine⁄ cysteine for 3 h and subjected to
immunoprecipitation using anti-TNSALP serum,
fol-lowed by SDS⁄ PAGE ⁄ fluorography as shown in
Fig 1 Under reducing conditions, the wild-type and
the two TNSALP mutants gave a similar
electropho-retic pattern, consisting of the 66 kDa and 80 kDa
forms However, strikingly, a distinct pattern was
obtained under nonreducing conditions In addition to
the two molecular forms, a 160 kDa and a 130 kDa
form were found only in the cells expressing
TNS-ALP(R433C) (Fig 1, lanes 2 and 6), indicating that a
considerable portion of newly synthesized
TNS-ALP(R433C) is covalently cross-linked via a disulfide
bond As reported previously [13], TNSALP(D289V) is
not processed to the 80 kDa form, as this mutant is
transport-incompetent (Fig 1, lanes 4 and 8) Instead
of being conveyed to the Golgi apparatus, it
accumu-lates in the ER, and is eventually degraded in the
ubiquitin–proteasome pathway [13] We consistently
observed a high molecular mass aggregate even in
the cells expressing the wild-type under nonreducing
conditions (see the top of the gel, Fig 1, lanes 5–8) Previously, we reported that a proportion of the newly synthesized TNSALP fails to be modified with GPI, and resultant GPI-anchorless TNSALP molecules form the aggregate in transfected cells [17] This probably reflects a shortage of a GPI precursor pool in the ER
of COS-1 cells where TNSALP is overexpressed ectopi-cally
The two TNSALP mutants appear
on the cell surface Next, we investigated whether the TNSALP mutants gain access to the cell surface like the wild-type The cells that expressed each TNSALP mutant were meta-bolically labeled and further incubated with phosphati-dylinositol-specific phospholipase C (PI-PLC) Upon digestion, the 80 kDa form was the only form in the culture media of the cells that expressed the wild-type
or TNSALP(R433H) (Fig 2, lanes 4 and 12) However, the 160 kDa form as well as the 80 kDa form were released into the medium from the cells expressing TNSALP(R433C) (Fig 2, lane 8), indicating that the dimerization via a disulfide bridge does not severely affect the cell surface appearance of TNSALP(R433C)
As a negative control, no TNSALP(D289V) was released into the medium by digestion with PI-PLC, because this mutant fails to exit from the ER Immuno-fluorescence studies also confirmed the cell surface appearance of the wild-type, TNSALP(R433C) and TNSALP(R433H), but not TNSALP(D289V) (data not shown)
Catalytic activity of TNSALP mutants
An immunoblotting method showed essentially the same result for steady-state expression of the TNSALP mutants as the biosynthetic experiments (Fig 3A, lanes
5 and 6), confirming that TNSALP(R433C) tends to become a disulfide-bonded form that is clearly differ-ent from the noncovaldiffer-ently associated forms of the wild-type, which migrates on the SDS gel as the
66 kDa or 80 kDa form
To address the question of whether the replacement
of arginine at position 433 affects the catalytic function
of TNSALP, the cells expressing the two mutants were assayed for alkaline phosphatase activity using p-nitro-phenylphosphate as a substrate (Fig 3B) Conservative replacement of arginine with histidine was expected not to greatly change the catalytic function of TNSALP(R433H), although we consistently detected higher specific enzyme activity in the cells expres-sing TNSALP(R433H) than in those expresexpres-sing the
a D k 6 a D k 3 a D k 8 a D k 6
8 7 6 5 4 3 2
1
d r n n d
r
Fig 1 Biosynthesis of TNSALP mutants in COS-1 cells COS-1
cells, which had been transfected for 24 h with a plasmid
enco-ding the wild-type (lanes 1 and 5), TNSALP(R433C) (lanes 2 and 6),
TNSALP(R433H) (lanes 3 and 7) or TNSALP(D289V) (lanes 4 and 8),
were labeled with [35S]methionine ⁄ cysteine for 3 h The cell lysates
were immunoprecipitated with anti-TNSALP, and the immune
com-plexes were then analyzed by SDS ⁄ PAGE ⁄ fluorography under
redu-cing (lanes 1–4) or nonreduredu-cing (lanes 5–8) conditions Double and
single arrowheads indicate the tops of the stacking and resolving
gels, respectively Left lane: 14 C-methylated protein markers of
200, 97.4, 66, 46 and 30 kDa, from the top of the gel.
Trang 4wild-type In contrast to this, the cell homogenate of the cells that expressed TNSALP(R433C) showed a much reduced level of activity as compared with the wild-type As a negative control, TNSALP(D289V) did not exhibit any enzyme activity, in agreement with a previous report [13] Km(Vmax) values for the wild-type, TNSALP(R433C) and TNSALP(R433H), which were determined using Lineweaver–Burk plots, were 0.23 mm (2.57 lmolÆmin)1), 0.50 mm (1.05 lmolÆmin)1) and 0.34 mm (3.69 lmolÆmin)1), respectively As the expression level of TNSALP(R433H) was higher than that of the wild-type in the COS-1 cells, based on the immunoblotting results (Fig 3A, lanes 1 and 3), it seems reasonable to assume that replacement of argin-ine with histidargin-ine at position 433 does not have much affect on the catalytic function of TNSALP, although a definite conclusion awaits its purification In the case of TNSALP(R433C), however, we were uncertain whether the decrease in specific enzyme activity could be attrib-uted to disulfide bond formation, as a significant amount of the noncross-linked molecular species was also present in the cell homogenate (Fig 3A, lane 6)
Expression of TNSALP(R433C) in CHO-K1 Tet-On cells
As it was difficult to separate the noncross-linked and the cross-linked form of TNSALP(R433C) from each other in the native state by means of biochemical methods such as gel filtration and electrophoresis, we turned to another strategy We reasoned that if expres-sion levels of TNSALP(R433C) are kept at a relatively low level compared with transient expression, most of the newly synthesized TNSALP(R433C) molecules might be oxidized to become disulfide-bonded in the
C L
M C M C M C M C M C M C M C M C
C 3 4 R d-type l
a D k 6 a D k 3 a D k 8 a D k 6
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
Fig 2 Cell surface appearance of TNSALP
mutants in COS-1 cells COS-1 cells, which
had been transfected with a plasmid
enco-ding the wild-type, TNSALP(R433C),
TNS-ALP(R433H) or TNSALP(D289V) for 24 h,
were labeled with [ 35 S]methionine ⁄ cysteine
for 3 h and chased for 2 h The cells were
then further incubated in the absence or
presence of PI-PLC The cell lysates (C) and
media (M) were immunoprecipitated with
anti-TNSALP, and the immune complexes
were analysed by SDS ⁄ PAGE
(nonreduc-ing) ⁄ fluorography The single arrowhead
indicates the top of the resolving gels Left
lane:14C-methylated protein markers as in
Fig 1.
0
0
0
0
0
0
0
0
0
0
0
V 9 2 D H 3 4 R C 3 4 R T W
B
4 3 2
d e n o n d
e
a D k 6 a D k 3 a D k 8 a D k 6
A
Fig 3 Steady-state expression of TNSALP mutants in COS-1 cells.
(A) COS-1 cells, which had been transfected with a plasmid
enco-ding the wild-type (lanes 1 and 5), TNSALP(R433C) (lanes 2 and 6),
TNSALP(R433H) (lanes 3 and 7) or TNSALP(D289V) (lanes 4 and 8)
for 24 h, were homogenized, and 10 lg of each homogenate was
directly separated by SDS ⁄ PAGE under reducing (lanes 1–4) or
non-reducing (lanes 5–8) conditions and subjected to immunoblotting
using anti-TNSALP Double and single arrowheads indicate the tops
of the stacking and resolving gels, respectively (B) The same
homogenates as described in (A) were assayed for alkaline
phos-phatase activity and protein Values are means of two independent
experiments.
Trang 5ER This was the case We succeeded in establishing a
CHO-K1 Tet-On (Tet-On) cell line that expresses
TNSALP(R433C) only in response to the addition of
doxycycline (a tetracycline analog) In marked contrast
to transient expression (Fig 3), the 160 kDa
disulfide-bonded form was the predominant molecular species
in the Tet-On cells, with a trace amount of the 80 kDa
noncross-linked form, over a wide range of expression
conditions (Fig 4) Induction of TNSALP(R433C)
was found to be regulated tightly, as no band was
observed in the absence of doxycycline (Fig 4)
Con-sistent with this, the alkaline phosphatase activity of
Tet-On cells was negligible in the absence of the indu-cer (data not shown) When its synthesis was induced, TNSALP(R433C) was localized on the cell surface of the Tet-On cells, as judged by immunofluorescence (Fig 5A) and PI-PLC digestion (Fig 5B) Next, the detergent extracts of cells expressing the wild-type or TNSALP(R433C) were fractionated by sucrose density gradient centrifugation, and the distribution of TNSALP was analyzed by immunoprecipitation (Fig 6) Both the wild-type and TNSALP(R433C) appeared at exactly the same position across the gradient, demonstrating that the disulfide-bonded
0.5 0.2
0
nonred red
a D k 0 1 a D k 0 1
a D k 8 a D k 6
Fig 4 Steady-state expression of TNS-ALP(R433C) in Tet-On cells.The established Tet-On cells harboring a plasmid encoding TNSALP(R433C) were cultured with differ-ent concdiffer-entrations of doxycycline for 24 h The cells were homogenized, and 5 lg of each homogenate was separated by SDS ⁄ PAGE under reducing (red) or non-reducing (nonred) conditions; this was fol-lowed by immunoblotting with anti-TNSALP DOX, doxycycline.
M C M C
a D k 6 a D k 3 a D k 8 a D k 6
Fig 5 Cell surface appearance of TNSALP(R433C) in Tet-On cells (A) Established Tet-On cells harboring a plasmid encoding TNS-ALP(R433C) were cultured with 0.5 lgÆmL)1doxycycline for 24 h After fixation, the cells were reacted with anti-TNSALP and then with anti-(rabbit IgG)–rhodamine (B) The established Tet-On cells, which had been cultured with 1.0 lgÆmL)1doxycycline for 14 h, were labeled with [ 35 S]methionine ⁄ cysteine for 0.5 h and chased for 1 h The cells were further incubated in the absence or presence of PI-PLC The cell lysates (C) and media (M) were immunoprecipitated with anti-TNSALP, and the immune complexes were analysed by SDS ⁄ PAGE (nonreduc-ing) ⁄ fluorography Left lane: 14 C-methylated protein markers as in Fig 1.
Trang 6TNSALP(R433C) forms a dimer like the wild-type.
The pulse-chase experiments demonstrated that the
wild-type 66 kDa form was efficiently processed to the
mature 80 kDa form, and this mature form was the only form found in the cell at 2 h chase time (Fig 7A) Similarly, the majority of TNSALP(R433C) was
12 11 10 9 8 7 6 5 4 3 2 1
Wild-type
C 3 4 R
a D k 8
a D k 6
a D k 8
c a b
Fig 6 Sucrose density gradient analysis of TNSALP(R433C) The established Tet-On cells harboring a plasmid encoding the wild-type or TNSALP(R433C) were cultured with 1.0 lgÆmL)1doxycycline for 12 h The cells were labeled with [35S]methionine ⁄ cysteine for 1 h and fur-ther chased for 3 h The cells were lysed, loaded on the top of the gradient [5–35% (w ⁄ w) sucrose], and centrifuged for 18 h at 4 C Each
400 lL fraction was collected from the top (fraction 1) of the gradient and immunoprecipitated The immune complexes were separated by SDS ⁄ PAGE (nonreducing), followed by fluorography The arrowhead indicates an unknown band BSA (b, 68 kDa), alcohol dehydrogenase (a,
141 kDa) and catalase (c, 250 kDa) were applied on a separate gradient as size markers Left lane: 14 C-methylated protein markers of 200, 97.4 and 66 kDa from the top of the gel.
a D k 8 a D k 6
a D k 6 a D k 3
a D k 6
0 ) h ( e a h C
2 1 5 0
e p y t d l W
C 3 4 R
a D k 3
a D k 6
+ -H o d E
Fig 7 Biosynthesis of TNSALP(R433C) in Tet-On cells (A) The established Tet-On cells harboring a plasmid encoding the wild-type or TNSALP(R433C) were cultured with 1.0 lgÆmL)1doxycycline for 14 h, labeled with [ 35 S]methionine ⁄ cysteine for 0.5 h, and chased for up to
2 h The cells were lysed and immunoprecipitated with anti-TNSALP, and the immune complexes were separated by SDS ⁄ PAGE (non-reducing), followed by fluorography Left lane: 14 C-methylated protein markers of 200, 97.4 and 66 kDa from the top of the gel (B) The established Tet-On cells harboring a plasmid encoding TNSALP(R433C) were cultured with 1.0 lgÆmL)1doxycycline for 14 h The cells were pulse-labeled with [ 35 S]methionine ⁄ cysteine for 0.5 h and immunoprecipitated for Endo H digestion The immunoprecipitates were analyzed
by SDS ⁄ PAGE (nonreducing) ⁄ fluorography.
Trang 7efficiently converted to the 160 kDa form, although a
small proportion of it remained unprocessed even after
2 h of chase Thus we cannot exclude the possibility
that this missense mutation also affects acquisition of
the transport competence of TNSALP The
dimeri-zation of TNSALP(R433C) must occur at the ER, as
the 130 kDa form appeared immediately after the
pulse period (Fig 7A) Furthermore, the 130 kDa
disulfide-bonded TNSALP(R433C) was sensitive to
Endo H digestion (Fig 7B)
The disulfide bridge suppresses the catalytic
function of TNSALP(R433C)
The predominance of the dimer form of
TNS-ALP(R433C) in the Tet-On cells in response to
doxy-cycline allowed us to unambiguously evaluate the
enzyme activity of this disulfide-bonded TNSALP
(R433C) (Fig 8) The specific enzyme activity of the
cells expressing TNSALP(R433C) was only
one-twen-tieth of those expressing the wild-type enzyme Km(and
Vmax) values obtained by kinetic studies are: 0.45 mm
(6.75 lmolÆmin)1) for the wild-type and 0.66 mm
(0.34 lmolÆmin)1) for the disulfide-bonded TNSALP
(R433C) As the wild-type and TNSALP(R433C) in
Tet-On cells were comparable in their expression levels
as estimated by immunoblotting (Fig 9, lanes 1 and 2),
it is likely that the disulfide bond formation
substan-tially suppresses the catalytic efficiency of
TNS-ALP(R433C) without much affecting its substrate
binding
Figure 9 shows the effect of dithiothreitol on the biosynthesis of TNSALP(R433C) Dithiothreitol is a membrane-permeable reducing agent and is known to render the lumen of the ER unfavorable for oxida-tion of sulfhydryl groups on cysteine residues [18] The cells were incubated with doxycycline in the absence or presence of dithiothreitol for 12 h or
24 h A small but significant amount of the 80 kDa form of TNSALP(R433C) was found to appear in the cells only with dithiothreitol (Fig 9A, lanes 3 and 7) A concentration of 1 mm of dithiothreitol was optimal, and higher concentrations of dithiothrei-tol tended to inhibit the synthesis of TNSALP (R433C) induced by doxycycline Importantly, we detected an increase in the enzyme activity of the cells concomitantly with the appearance of the
80 kDa form (Fig 9B), suggesting that TNSALP (R433C) is capable of exhibiting its catalytic activity unless it is oxidized to form an interchain disulfide bond However, we failed to increase the enzyme activity of the cell homogenate prepared from Tet-On cells expressing TNSALP(R433C) by incubating them with dithiothreitol or 2-mercaptoethanol under var-ious conditions
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
1
C 3 4 R T
W
Fig 8 Alkaline phosphatase activity in the Tet-On cells expressing
TNSALP(R433C) After the established Tet-On cells harboring a
plasmid encoding the wild-type or TNSALP(R433C) had been
cul-tured with 1 lgÆmL)1doxycycline for 24 h, the cells were
homo-genized and assayed for alkaline phosphatase and protein The
homogenates (5 lg each) were also used for immunoblotting
(Fig 9, lanes 1 and 2) Values are means of two independent
experiments.
Fig 9 Effects of dithiothreitol on the expression of TNS-ALP(R433C) After the established Tet-On cells harboring a plasmid encoding the wild-type (lane 1) or TNSALP (R433C) (lanes 2–9) had been cultured with 1 lgÆmL)1doxycycline for 12 h or 24 h in the presence of different concentrations of dithiothreitol (A), the cell homogenates (5 lg each) were used for immunoblotting (nonreduc-ing) (B) The same cell homogenates as described in (A) were investigated for alkaline phosphatase activity The open bar and closed bar represent 24 h or 12 h of incubation with dithiothreitol, respectively Values are means of two experiments.
Trang 8Next, we compared protease susceptibility between
the wild-type and TNSALP(R433C) As shown in
Fig 10, the wild-type enzyme was largely resistant to
trypsin digestion at concentrations up to 50 lgÆmL)1,
whereas the mutant protein was found to be degraded
at higher concentrations of trypsin The same holds
true for proteinase K digestion The mutant protein
completely disappeared even at 0.5 lgÆmL)1 (lane 7),
but not the wild-type (lane 2) These results therefore
suggest that the interchain disulfide bond markedly
changes the tertiary structure of TNSALP such that
TNSALP(R433C) becomes more susceptible to the
proteases
An interchain disulfide bridge forms between
two cysteines at position 433
Human TNSALPs have five cysteine residues (C102,
C122, C184, C472 and C480) per subunit, and their
positions are well conserved among four isoenzymes
[3,19] C122 and C472 are thought to bond to C184
and C480 in the same subunit, respectively, whereas
C102 is in a free state, raising the possibility that
C102 is involved in the interchain disulfide bridge of
TNSALP(R433C) To address this question, we
replaced C102 with serine and expressed TNSALP
(C102S) in the COS-1 cells as shown in Fig 11
TNSALP(C102S) consists of the 66 kDa immature and
80 kDa mature forms, and showed a similar specific
enzyme activity to that of the wild-type Also, a
TNS-ALP double mutant (C102S⁄ R433C) was found to be
indistinguishable from TNSALP(R433C) as assessed
by immunoblotting, as shown in Fig 11A (lanes 7 and 8), suggesting that a disulfide bond forms between C433 residues on two subunits of TNSALP(R433C)
Discussion
Hypophosphatasia and TNSALP mutants Inorganic pyrophosphate is believed to play a pivotal role in bone matrix mineralization [20,21] At lower concentrations (0.01–0.1 mm), pyrophosphate enhances mineralization, whereas it inhibits the formation of hydroxyapatite at concentrations higher than 1 mm TNSALP is thought to promote mineralization by hydrolyzing pyrophosphate into phosphate Fine regu-lation of pyrophosphate levels at the site of mineral-ization also requires at least two other proteins: nucleoside triphosphate pyrophosphatase phospho-diesterase (or PC-1), which generates pyrophosphate from nucleoside triphosphate, and a channel protein ANK (ankylosis), which mediates transport of pyro-phosphate across the plasma membrane of osteoblasts [3,22,23]
Various mutations in the TNSALP gene cause a her-editary disease known as hypophosphatasia, which is characterized by defective osteogenesis, unequivocally pointing to the physiologic relevance of the enzyme in biomineralization [4,5,7] In support of this, relevant knock-out mice develop rickets and osteomalacia, thus recapitulating infantile hypophosphatasia [24–26] In TNSALP-deficient mice, the initiation of mineral crystallization occurs within matrix vesicles; however,
2 1 0
9 8 7 6 5 4 3 2
C 3 4 R d-type
l w C
3 4 R d-type
l w
n i s p y r
a D k 0
a D k 0 1
Fig 10 Protease sensitivity After the established Tet-On cells harboring a plasmid encoding the wild-type or TNSALP(R433C) had been cul-tured with 1 lgÆmL)1doxycycline for 24 h, the cells were homogenized in 10 m M Tris ⁄ HCl (pH 8.0), using a sonicator The homogenates were incubated with trypsin or proteinase K in an ice ⁄ water bath for 30 min at the indicated concentrations For trypsin, the final concentra-tions were (lg ⁄ mL): lanes 1 and 6, 0; lanes 2 and 7, 5; lanes 3 and 8, 10; lanes 4 and 9, 20; lanes 5 and 10, 50 For proteinase K, the final concentrations were (lg ⁄ mL): lanes 1 and 6, 0; lanes 2 and 7, 0.5; lanes 3 and 8, 1.0; lanes 4 and 9, 5.0; lanes 5 and 10, 10 Lanes 1–5 and lanes 6–10 were analyzed by SDS ⁄ PAGE in the presence or absence of 2-mercaptoethanol, respectively.
Trang 9the subsequent proliferation and growth stage of
min-eralization is severely impaired, leading to an increase
in noncalcified bone matrix (osteoid) [27], consistent
with what is seen in hypophosphatasia patients [28]
Hypophosphatasia patients show wide-ranging clinical
manifestations, from stillbirth with an almost
unminer-alized skeleton to premature loss of deciduous teeth in
childhood and pseudofracture first presenting in adult
life [1,2] The symptoms of hypophosphatasia are well
known to correlate with the residual enzyme activities
of affected patients [1,2,6,7] During the course of our
studies on the biosynthesis of several TNSALP
mutants, we found that the missense mutations
associ-ated with severe hypophosphatasia variously affect the
efficiency with which TNSALP properly folds and
cor-rectly assembles, depending upon the position of a
missense mutation and the nature of a substituted
amino acid For example, TNSALP(A162T), found in
a homozygous patient diagnosed with a lethal infantile
form of hypophosphatasia [4], mainly formed a high
molecular mass aggregate in the ER, and only a small
proportion of newly synthesized TNSALP(A162T)
reached its site of action, the cell surface [9,10]
Conse-quently, the cell surface expression of this TNSALP
mutant is much reduced compared with that of the
wild-type More TNSALP(D277A) was found to gain
access to the cell surface than TNSALP(A162T),
albeit with a significant population in the ER [10]
Alternatively, TNSALP(R54C), TNSALP(N153D),
TNSALP(E218G), TNSALP(D289V) and TNSALP
(G317D) never appeared on the plasma membrane
[9–13] Interestingly, a recent study has shown that
alkaline phosphatase acquires Zn2+, which is indis-pensable for its catalytic activity, in the Golgi apparatus on its way to the plasma membrane [29] This leads to the speculation that TNSALP mutants, which are retained in the ER due to a folding defect, not only fail to appear on the cell surface, but also are not able to acquire Zn2+ Consistent with this, the TNSALP mutants with defective ER-to-Golgi transport did not show measurable alkaline phospha-tase activity when being expressed in COS-1 cells [10–13]
TNSALP(R433C) becomes cross-linked via
a disulfide bridge Mammalian alkaline phosphatases have five cysteine residues per subunit, and their positions are well con-served [3,19,30] C102 is believed to be present only
in a free state, whereas C122 and C472 bind to C184 and C480, respectively, in the same subunit Both TNSALP(C184Y) and TNSALP(C472S) have been reported in perinatal hypophosphatasia patients [15,31], implying that the two interchain disulfide bonds are necessary for the correct folding and assembly of TNSALP TNSALP(R433C) was repor-ted in homozygous patients diagnosed with lethal infantile hypophosphatasia [16,32] In contrast to the TNSALP mutants showing various degrees of folding defect, TNSALP(R433C) did not form a high molecu-lar mass aggregate Instead, it formed a covalently cross-linked homodimer, as evidenced by sucrose den-sity gradient centrifugation (Fig 6) As replacement
reducing
6000
5000
4000
160 kDa
80 kDa
vity (U/mg protein) 3000 2000
1000
0
WT C102S R433C C102S/R433C
Fig 11 Expression of a TNSALP double mutant (C102S ⁄ R433C) in COS-1 cells COS-1 cells expressing wild-type enzyme (lanes 1 and 5), TNSALP(C102S) (lanes 2 and 6), TNSALP(R433C) (lanes 3 and 7) or TNSALP(C102S ⁄ R433C) (lanes 4 and 8) were homogenized The homo-genates were analyzed by SDS ⁄ PAGE under reducing or nonreducing conditions, and this was followed by immunoblotting with anti-TNSALP (A) or assayed for alkaline phosphatase (B).
Trang 10of C102 with serine did not affect the cross-linking of
TNSALP(R433C) (Fig 11), this result strongly
indi-cates that a sulfhydryl group on the cysteine residue
at position 433 of one subunit is oxidized to bond to
the counterpart of the other subunit This covalent
cross-linkage of TNSALP(R433C) occurs in an early
stage of the secretory pathway, as the cross-linked
molecular species appeared in the cell immediately
after a pulse-labeling period, and besides this, the
130 kDa form was sensitive to Endo H digestion
Also, the results of the pulse-chase experiments
suggest that most newly synthesized TNSALP(R433C)
migrated from the ER to the Golgi apparatus at a
similar rate to the wild-type enzyme (Fig 7) The cell
surface appearance of TNSALP(R433C) was shown
by immunofluorescence microscopy and PI-PLC
digestion (Fig 5), indicating that TNSALP(R433C)
resides on the cell surface as a GPI-anchored
ecto-enzyme, like the wild-type Thus, it is likely that the
cross-linkage between the subunits did not greatly
affect the biosynthesis and intracellular transport of
this mutant protein However, the intersubunit
cross-linkage did severely affect the catalytic activity of
TNSALP(R433C) This is based on the findings in
Tet-On cells, which predominantly express the
cross-linked form of TNSALP(R433C) in response to
doxy-cycline Considering that the expression levels of
TNSALP(R433C) and the wild-type in each Tet-On
cell line are very similar (Fig 9A), comparison of Km
and Vmax values suggests that the catalytic efficiency
of the mutant protein is dramatically reduced
com-pared with that of the wild-type Increased
suscepti-bility of TNSALP(R433C) to proteases supports the
notion that the disulfide bridge has a profound effect
on the structure of TNSALP (Fig 10) The effects of
substitution of R433 either with alanine or aspartate
on the catalytic properties of TNSALP were reported
by Kozlenkov et al [33] Both TNSALP(R433A) and
TNSALP(R433D) showed a noticeable decrease in
kcat with a moderate increase in Km One might argue
that the substitution of arginine with cysteine itself,
but not the disulfide bridge, decreases the catalytic
activity of TNSALP(R433C) However, this is
unli-kely, for the following reasons: first, the COS-1 cells,
which express both noncross-linked and cross-linked
TNSALP(R433C), showed considerable enzyme
acti-vity (Fig 3) Second, when the Tet-On (R433C) cells
were cultured in the presence of doxycycline and
di-thiothreitol, a significant amount of TNSALP(R433C)
failed to become cross-linked, and concomitantly we
detected an increase in enzyme activity in the cell
homogenates Taken together, these facts suggest that
the diminished catalytic function of TNSALP(R433C)
due to its disulfide-bonded linkage is a likely cause for the lethal hypophosphatasia resulting from the homozygous presence of this mutation To our know-ledge, this is the first TNSALP missense mutation associated with severe hypophosphatasia that abro-gates the catalytic activity of TNSALP without signi-ficantly affecting its cell surface expression
TNSALP(R433H) was reported in a compound heterozygote (R433H⁄ D389G) diagnosed with a mild form of hypophosphatasia [15] Therefore, it is reason-able to assume that TNSALP(R433H) does not have a severe effect, unlike TNSALP(R433C) Also, as the substitution of arginine with histidine is a conservative replacement, it was expected that this mutation would not much affect TNSALP activity When expressed in COS-1 cells, TNSALP(R433H) showed enzyme activity comparable to that of the wild-type Also, its biosyn-thesis and cell surface appearance were not measurably disturbed (Figs 1–3), further highlighting the clinical importance of the substitution of arginine at position
433 with cysteine
Experimental procedures
Materials
chemiluminescence western blotting detection reagent, per-oxidase-conjugated donkey anti-(rabbit IgG) and protein A–Sepharose CL-4B were obtained from Amersham Phar-macia Biotech (Arlington Heights, IL); the pALTER-MAX,
obtained from Promega (Madison, WI); the QuikChange II Site-Directed Mutagenesis kit was obtained from Stratagene (La Jolla, CA); G418 and pansorbin were obtained from Calbiochem (La Jolla CA); Lipofectamine Plus Reagent was obtained from Invitrogen (Carlsbad, CA); PI-PLC was obtained from BIOMOL International, L.P (Plymouth Meeting, PA); aprotinin, doxycycline and saponin (Quillaja
ketone-treated bovine pancreas trypsin were obtained from Sigma Chemical Co (St Louis, MO); proteinase K was obtained from Roche Diagnotics (London, UK); antipain, chymostatin, elastatinal, leupeptin and pepstatin A were obtained from the Protein Research Foundation (Osaka, Japan); hygromycin B and p-amidinophenylmethanesulfonyl fluoride were obtained from Wako Pure Chemicals (Tokyo, Japan); and serum against recombinant human TNSALP was raised in rabbits as described previously [34] pTRE2 and the BD CHO-K1 Tet-On cell line and Tet system approved fetal bovine serum were obtaied from BD Biosciences Clontech (Palo Alto, CA)