Tài liệu Báo cáo khoa học: Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension pdf

In support of this, alkaline phosphatase activity of the cells expressing TNS-ALP 1559delT was localized at the juxtanucleus position, but not on the cell surface.. Thus, we conclude tha

of tissue-nonspecific alkaline phosphatase is ascribed

to three cysteine residues in the C-terminal extension

Retarded secretion and proteasomal degradation

Keiichi Komaru1,2, Yoko Ishida1, Yoshihiro Amaya1, Masae Goseki-Sone3, Hideo Orimo4

and Kimimitsu Oda1,5

1 Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Gakkocho-dori, Niigata, Japan

2 Kitasato Junior College of Health and Hygienic Sciences, Yamatomachi, Minami-Uonuma-shi, Niigata, Japan

3 Department of Food and Nutrition, Japan Women’s University, Mejirodai, Bunkyo-ku, Tokyo, Japan

4 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan

5 Center for Transdisciplinary Research, Niigata University, Japan

Keywords

aggregation; alkaline phosphatase;

degradation; hypophosphatasia; proteasome;

ubiquitin

Correspondence

K Oda, Division of Biochemistry, Course for

Oral Life Science, Niigata University,

Graduate School of Medical and Dental

Sciences, 2–5274, Gakkocho-dori, Niigata,

951–8514, Japan

Fax: +81 25 227 2831

Tel: +81 25 227 2827

E-mail: oda@dent.niigata-u.ac.jp

(Received 21 December 2004, revised 30

January 2005, accepted 3 February 2005)

doi:10.1111/j.1742-4658.2005.04597.x

In the majority of hypophosphatasia patients, reductions in the serum lev-els of alkaline phosphatase activity are caused by various missense muta-tions in the tissue-nonspecific alkaline phosphatase (TNSALP) gene A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency In this study, we examined the molecular phenotype

of TNSALP (1559delT) using in vitro translation⁄ translocation system and COS-1 cells transiently expressing this mutant protein We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by  12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor In support of this, alkaline phosphatase activity of the cells expressing TNS-ALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface However, only a limited amount of the newly synthesized pro-tein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome SDS⁄ PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms

a disulfide-bonded high-molecular-mass aggregate Interestingly, the aggre-gate form of TNSALP (1559delT) exhibited a significant enzyme activity When all three cysteines at positions of 506, 521 and 577 of TNS-ALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associ-ated dimer, strongly indicating that these cysteine residues in the C-ter-minal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT)

Abbreviations

Bz-Asn-Gly-Thr-NH2, benzoyl-asparagine-glycine-threonine-amide; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; ECL, enhanced chemiluminescence; GPI, glycosylphosphatidylinositol; LLnL, N-acetyl- L -leucinyl- L -leucinyl- L -norleucinal); LLM, N-acetyl- L -leucinyl- L -leucinyl- L -methional); MG-132, benzyloxycarbonyl- L -leucinyl- L -leucinyl- L -leucinal; PI-PLC, phosphatidylinositol-specific phospholipase C; PNGase F, peptide:N-glycosidase F; MEM, minimum essential medium; TNSALP, tissue-nonspecific alkaline phosphatase; sTNSALP, soluble form of TNSALP.

Hypophosphatasia is an inborn error of metabolism

characterized by defective mineralization of hard

tis-sues and reduced levels of tissue nonspecific alkaline

phosphatase (TNSALP, EC 3.1.3.1) [1–3]

Hypophos-phatasia is classified into at least five categories

depending on age of onset and severity: perinatal,

infantile, childhood, adult and

odontohypophosphata-sia The disease is caused by various mutations in the

TNSALP gene, which is located on chromosome

1p-36.1–34, and is transmitted in an autosomal

reces-sive or a dominant manner The severity of the disease

is inversely related to serum levels of alkaline

phospha-tase activity, therefore indicating that reduction of

enzyme activity caused by the defective TNSALP genes

are responsible for poor mineralization of bone and

tooth

TNSALP-deficient mice develop rickets and

osteope-nia postnatally and many die of seizure [4–6],

recapit-ulating infantile hypophosphatasia Additionally, as

with hypophosphatasia patients, elevated levels of

inor-ganic pyrophosphate, phosphoethanolamine and

pyrid-oxal-5¢-phosphate have been reported in the serum and

urine of the knockout mice This raises the possibility

that these phosphocompounds are natural substrates

for TNSALP Recently Hessle et al [7] have postulated

that the concerted action of nucleoside triphosphate

pyrophosphohydolase and TNSALP regulates local

concentration of inorganic pyrophosphate at the site of

mineralization, which, as a poison of hydroxyapatite

growth, in turn controls mineralization According to

this idea, increased levels of inorganic pyrophosphate

resulting from the defect of TNSALP are thought to

be the main cause of hypomineralization

Since Weiss et al [8] first identified a missense

muta-tion (Ala162Thr) in the TNSALP gene of a patient

diagnosed with infantile hypophosphatasia, more than

161 mutations have been reported and of all mutations

about 80% of them are missense [3,9,(http://www.sesep

uvsq.fr/Database.html)] To date, biochemical

charac-terization of TNSALP mutant proteins have been

lim-ited to a small number of cases Nevertheless, some

missense mutations, in particular associated with severe

forms of hypophophatasia, were known to impair

proper folding and correct assembly of TNSALP in the

endoplasmic reticulum (ER), resulting in the loss of

functional TNSALP from the cell surface [10–14]

Dele-tion of T at posiDele-tion 1559 of the cDNA

[TNS-ALP (1559delT)] of the TNS[TNS-ALP gene was reported

for the first time in hypophosphatasia patients by Orimo

et al [15] and TNSALP (1559delT) is transmitted as a

recessive trait TNSALP (1559delT) was originally

referred to as TNSALP (1735delT) [16–18] According

to the recommended nomenclature, the adenine of the

initiator ATG codon in the cDNA of TNSALP is denoted as nucleotide +1 instead of the first nucleotide

of the cDNA clone originally isolated by Weiss et al [8] TNSALP (1559delT) is unique in that so far, this frameshift mutation has been reported only in the Jap-anese population [18] The patients are largely com-pound heterozygotes with different missense mutations,

or in some cases with undetected mutations on the opposite allele, and exhibit clinical manifestations vary-ing from infantile to odontohypophosphatasia [18] Quite recently, a case of a homozygous patient of TNS-ALP (1559delT) has been reported and classified as the prenatal lethal form, confirming that this mutation rep-resents a severe allele [19] This frameshift mutation

is assumed to eliminate the original translational stop codon and instead causes the extension consisting of 80 amino acid residues at the C-terminus of TNSALP Goseki et al detected a larger form of TNSALP in vivo

in the serum of the patients carrying this mutation [16]; however, little is known about the molecular phenotype

of TNSALP (1559delT) underlying the clinical symp-toms

In this report we have elucidated the biosynthesis

of TNSALP (1559delT) in a heterologous expression system and in vitro translation⁄ translocation system Our results shows that although TNSALP (1559delT)

is synthesized as a secretory form lacking glycosylphos-phatidylinositol (GPI), most of newly synthesized mole-cules form the aggregate and fail to exit from the ER Furthermore, the accumulated TNSALP (1559delT) was found to be polyubiquitinated under the condition where cellular proteasome activity was blocked, indicat-ive of ubiquitin⁄ proteasome pathway as part of an ER quality control mechanism We also have demonstrated that three cysteine residues in the C-terminal extension

of this frameshift mutant protein are responsible for the formation of the novel aggregate retaining enzyme activity

Results

In vitro translation/translocation The deletion of T at cDNA number 1559 causes a frameshift downstream from leucine at position 503 of TNSALP, resulting in the elimination of an original translational stop codon Thus, the cDNA of TNS-ALP (1559delT) was predicted to encode a large sized TNSALP molecule with an additional 80 amino acid-long extension at the C-terminus (Fig 1) To confirm this prediction, we performed in vitro translation experiments as shown in Fig 2A The molecular mass

of TNSALP (1559delT) was estimated to be 66 kDa

and larger than the wild type enzyme by  12 kDa.

This value is in close agreement with the calculated

molecular mass (65 796) based on the amino acid

sequence of TNSALP (1559delT) When translation was carried out in the presence of the canine micro-some, TNSALP (1559delT) became a 80 kDa form; however, the appearance of the 80 kDa form was greatly diminished in the presence of

Bz-Asn-Gly-Thr-NH2, an inhibitor of N-glycosylation (Fig 2A, lane 6) Furthermore, upon incubation with PNGase F, which cleaves N-linked oligosaccharides between innermost N-acetylglucosamine and asparagine residue of glyco-proteins, the 80 kDa form was completely converted

to the 66 kDa form (Fig 2B), indicating that TNS-ALP (1559delT) is cotranslationally N-glycosylated to become the 80 kDa form in the microsome Thus, it is unlikely that the additional 80 amino acid residues

at the C-terminus strongly affect the cotranslational translocation of TNSALP (1559delT) across the ER membrane

Phase separation using Triton X-114 Given our finding that TNSALP (1559delT) is synthes-ized as a larger protein with a C-terminal extension, we considered the possibility that this frame-shift mutant protein fails to be attached by a GPI, because a puta-tive GPI-anchor signal consisting of a stretch of hydro-phobic amino acids is abrogated Previous studies have demonstrated that the wild type TNSALP expressed in the COS-1 cell is modified by GPI as shown by its sen-sitivity to phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves between phosphatidylglycerol and phosphoinositol of GPI, and metabolic labeling using [3H]ethanolamine, a component of GPI [10–13]

To examine if TNSALP (1559delT) is modified by GPI,

we exploited a phase separation method by Bordier [20] After metabolic labeling, the cells expressing either the wild type or TNSALP (1559delT) were lysed in a buffer containing TX-114 on ice, then warmed at

25
C A detergent phase was separated from an aque-ous phase by centrifugation and both phases were sub-jected to immunoprecipitation Newly synthesized wild type enzyme was largely partitioned into the detergent phase as shown in Fig 3 (lanes 1 & 2) The band in the aqueous phase probably represents GPI-anchor-less molecules due to overexpression of the enzyme in the transiently transfected cells It is noteworthy that a

66 kDa form, but not an 80 kDa form was partitioned into the aqueous phase As the 66 kDa form of the wild type migrates to the Golgi complex and becomes the

80 kDa form as described previously [10,12], this result suggests that the GPI-less molecules fail to exit the

ER However, this partition behavior of the wild type enzyme completely changed upon incubation with PI-PLC prior to phase separation All wild type

80 kDa

80 kDa

66 kDa

66 kDa

54 kDa

54 kDa

1 2 3 4 5 6

A

B

Fig 2 In vitro transcription ⁄ translation (A) Transcription-coupled

translation of TNSALP or TNSALP (1559delT) were carried out in

the absence (lanes 1 and 4) or presence (lanes 2, 3, 5 and 6) of

canine pancreatic microsomes The N-glycosylation inhibitor

Bz-Asn-Gly-Thr-NH 2 was added at a final concentration of 0.5 m M

(lanes 3 and 6) Aliquots of the translation reactions were analysed

by SDS ⁄ PAGE, followed by fluorography The leftmost lane shows

14 C-methylated protein markers of 200, 97.4, 66 and 46 kDa, from

the top of the gel (B) The translation products (A, lanes 2 and 5)

were further incubated in the absence or presence of PNGase F,

followed by SDS ⁄ PAGE ⁄ fluorography Left lane: 14 C-methylated

protein markers as in Fig 2A.

TNSALP GPLLLALALYPLSVLF

506 521

1559delT GPLLLALALYP RASCSEGPGPGHPQARDRCQLPTRQPPSQGARWGPP

LQLQERGPRKPKSAAHLAPLWNLPQGPNPLLASSLCSLPAALWPTG

Fig 1 Predicted amino acid sequence of TNSALP (1559delT) A

single T deletion in the cDNA at nucleotide 1559 of tissue

nonspe-cific alkaline phosphatase (TNSALP) changes the amino acid

sequence at leucine 503 and downstream until the new stop codon

appears Accordingly, this frame-shift mutation predicts that

ALP (1559delT) is 80 amino acids longer than the wild type

TNS-ALP Three cysteine residues at positions of 506, 521 and 577 are

marked.

TNSALP molecules were now partitioned into the

aqueous phase (Fig 3, lanes 3 & 4), indicating that the

wild type TNSALP molecule in the detergent phase

represents a GPI-anchored membrane form In contrast

to the wild type, TNSALP (1559delT) was exclusively

found in the aqueous phase even without PI-PLC

diges-tion (Fig 3, lanes 7 & 8), strongly arguing that

TNSALP (1559delT) lacks a GPI For comparison, we

also expressed and analyzed a soluble truncated form

of TNSALP (sTNSALP), which lacks the C-terminal

23 amino acids including a putative GPI-anchor signal

sequence [21] As expected, sTNSALP was found to be

recovered only in the aqueous phase like TNSALP

(1559delT) (Fig 3, lanes 5 and 6), further

support-ing that TNSALP (1559delT) is not modified by a GPI

Biosynthesis of TNSALP (1559delT)

If TNSALP (1559delT) is not attached by a

GPI-anchor, a prediction is that this mutant is no longer

embedded into the lipid bilayer via GPI, which helps

anchor TNSALP to the plasma membrane, but is

secreted out of the cell To investigate whether this

mutant protein is secreted, we labeled the transfected

cells with [35S]methionine⁄ cysteine and followed the

kinetics of TNSALP secretion As reported previously

[10,12], the wild type TNSALP was synthesized as the

66 kDa Endo H-sensitive form and underwent

process-ing of N-linked oligosaccharides to become the 80 kDa

Endo H-resistant mature species Both the 66 kDa and

80 kDa forms of TNSALP were detected in the trans-fected cells, though the conversion of the precursor to the 80 kDa form obviously is not efficient in our tran-sient expression system (Fig 4A, lanes 1–3) In con-trast, in the cells expressing TNSALP (1559delT), an

80 kDa form – which corresponds in molecular mass

to the in vitro 80 kDa translational product (Fig 2) – was the only molecular species throughout the chase time (Fig 4A, lane 6) The intensity of the 80 kDa form of TNSALP (1559delT) rapidly decreased as the chase time elapsed However, this decline is not simply accounted for by the secretion of the mutant protein into the medium, as no band was detectable even in the 6 h chase culture medium (Fig 4A, lane 8) Only after a prolonged exposure, however, a 90 kDa form

of TNSALP (1559delT) was found in the medium (results not shown) To confirm that TNSALP (1559delT) is indeed secreted into the medium, albeit

in a lesser amount, culture media were collected from continuously radiolabeled transfected cells and any secreted TNSALP (1559delT) was immunoprecipitated

A 90 kDa form of TNSALP (1559delT) became evi-dent in the medium (Fig 4B, lane 4), suggesting that the 80 kDa form was processed to the 90 kDa form

in the Golgi apparatus before being released into the medium In support of this, this secretory form was found to be sensitive to PNGase F but resistant to Endo H (Fig 4B, lanes 5 & 6) On the other hand, the GPI-anchor-less sTNSALP was efficiently secreted out

of the cells even after 0.5 h chase (Fig 4C), indicating that sTNSALP behaves like a genuine secretory pro-tein Thus, we conclude that TNSALP (1559delT) is newly synthesized as the 80 kDa soluble form and mostly undergoes degradation, resulting in only a por-tion of it being secreted as the 90 kDa Endo H-resist-ant form

Degradation and ubiquitination of TNSALP (1559delT)

We next examined the effect of several protease inhibitors on the degradation of TNSALP (1559delT) Inhibitors of proteasome function, such as LLnL and MG-132, but not a calpain inhibitor (LLM) remark-ably blocked the degradation of TNSALP (1559delT) (Fig 5A,B), indicative of involvement of the protea-some Consistent with this observation, leupeptin and pepstatin A (inhibitors of lysosomal proteases) had no effect on the degradation (results not shown) Quite recently we have reported that TNSALP (D289V), which is associated with perinatal hypophosphatasia, undergoes polyubiquitination prior to the degradation

in the proteasome in the transfected COS-1 cells This

det aq det aq det aq det aq

TNSALP sTNSALP 1559delT

80 kDa

66 kDa

1 2 3 4 5 6 7 8

Fig 3 Phase separation COS-1 cells expressing the wild type

TNS-ALP (lanes 1–4), sTNSTNS-ALP (lanes 5 and 6) or TNSTNS-ALP (1559delT)

(lanes 7 and 8) were labeled with [ 35 S]methionine ⁄ cysteine for 3 h.

The cells were lysed in buffer containing Triton X-114 and

parti-tioned into detergent (det) and aqueous (aq) phases before (lanes 1,

2, 5–8) or after (lanes 3 and 4) PI-PLC treatment Each phase was

subjected to immunoprecipitation The immune complexes were

analysed by SDS⁄ PAGE, followed by fluorography Left lane: 14

C-methylated protein markers of 97.4, 66 and 46 kDa.

1 2 3 4 5 6 7 8

TNSALP 1559delT TNSALP 1559delT

cell medium

80 kDa

66 kDa

cell medium

1 2 3 4 5 6

80 kDa

90 kDa

54 kDa

cell medium

72 kDa

1 2 3 4 5 6 7

1559delT

sTNSALP

A

B

C

Fig 4 Pulse-chase experiment (A) Cells expressing TNSALP (lanes

1–3, 7) or TNSALP (1559delT) (lanes 4–6, 8) were pulse-labeled

with [ 35 S]methionine ⁄ cysteine for 30 min (lanes 1 and 4) and

chased for 3 h (lanes, 2 and 5) or for 6 h (lanes 3 and 6) At 6 h

chase period, the media (lanes 7 and 8) were removed and the

cells were lysed for immunoprecipitation The immune complexes

were analysed by SDS ⁄ PAGE and fluorography Left lane:

14 C-methylated protein markers of 97.4, 66 and 46 kDa (B) Cells

expressing TNSALP (1559delT) were labeled for 6 h with

[ 35 S]methionine ⁄ cysteine After 6 h, the medium was removed

(lanes 4–6) and the cells (lanes 1–3) were lysed for

immunoprecipi-tation The immunoprecipitates were incubated in the absence

(lanes 1 and 4) or presence of PNGase F (lanes 2 and 5) or Endo H

(lanes 3 and 6) prior to SDS ⁄ PAGE ⁄ fluorography Left lane:

14 C-methylated protein markers of 97.4, 66 and 46 kDa (C) Cells

expressing sTNSALP were pulse-labeled with [ 35 S]methionine ⁄

cys-teine for 30 min and chased for 0 h (lane 1), for 0.5 h (lanes 2 and

5), for 1 h (lanes 3 and 6) or 2 h (lanes 4 and 7) The media and cell

lysates were subjected to immunoprecipitation and the immune

complexes were analysed by SDS ⁄ PAGE ⁄ fluorography Left lane:

14 C-methylated protein markers of 200, 97.4, 66, 46 and 30 kDa.

Control LLM LLnL MG132

1 2 3 4 5 6 7 8 -LLnL +LLnL

80 kDa

80 kDa

1 2 3 4 5 6

HA-Ub - - + + - - + + LLnL - + - + - + - +

anti-Ub anti-HA

PolyUb

A

B

C

Fig 5 Degradation and ubiquitination (A) Cells expressing TNS-ALP (1559delT) were pulse-labeled with [35S]methionine ⁄ cysteine for 30 min and chased for 6 h in the absence (lanes 1 and 2) or presence of 50 l M LLM (lanes 3 and 4), 50 l M LLnL (lanes 5 and 6) or 50 l M MG-132 (lanes 7 and 8) The cell lysates were subjec-ted to immunoprecipitation and the immune complexes were ana-lysed by SDS ⁄ PAGE and fluorography Left lane: 14 C-methylated protein markers of 200, 97.4, 66, 46 and 30 kDa (B) Cells expres-sing TNSALP (1559delT) were pulse-labeled with [35 S]methion-ine ⁄ cysteine for 30 min and chased for 0 h (lanes 1 and 4), 3 h (lanes 2 and 5) or 6 h (lanes 3 and 6) in the absence (lanes 1–3) or presence of 50 l M LLnL (lanes 4–6) The immune complexes were analysed by SDS ⁄ PAGE and fluorography Left lane: 14 C-methylated protein markers (Fig 4A) (C) Cells expressing TNSALP (1559delT) alone or TNSALP (1559delT) and HA-ubiquitin were incubated in the absence (–) or presence (+) of 50 l M LLnL for 6 h Then the cells were lysed and subjected to immunoprecipitated with anti-TNSALP After transfer, membranes were reacted with antiubiquitin (anti-Ub) or anti-influenza hemagglutinin epitope (anti-HA) Igs.

finding prompted us to determine if TNSALP

(1559delT) also is ubiquitinated prior to degradation

in the proteasome To this end we transfected the cells

with the plasmid encoding TNSALP (1559delT) with

or without the plasmid encoding ubiquitin bearing the

N-terminal influenza HA epitope TNSALP (1559delT)

was immnoprecipitated with anti-TNSALP Igs and

subsequently the immunoprecipitates were subject to

immunoblotting using either anti-ubiquitin or anti-HA

Igs (Fig 5C) Not only did proteasome inhibitors did

not affect ubiquitination, but also overall biosynthesis

of the wild type enzyme (results not shown) [14]

Remarkably, TNSALP (1559delT) was found to be

heavily ubiquitinated in the presence of the inhibitor

of proteasome function Furthermore, the extent of

ubiquitination of TNSALP (1559delT) was further

augmented in the cells expressing ubiquitin, strongly

demonstrating that this mutant protein is degraded via

ubiquitin⁄ proteasome pathway

Catalytic activity of TNSALP (1559delT)

Figure 6 shows cytohistochemistry for alkaline

phos-phatase In contrast to the wild type enzyme, virtually

no alkaline phosphatase activity was detected on the

cell surface of cells expressing TNSALP (1559delT) A

faint staining might be attributed to secreted

TNS-ALP (1559delT) trapped on the cell surface because of

its aggregate nature (see below)

These observations are compatible with the finding

that the wild type, but not the mutant, protein is

attached by GPI as shown in Fig 2 Interestingly, we

detected strong alkaline phosphatase activity at a

juxtanucleus position in the cells expressing TNSALP

(1559delT) as well as the wild type, suggesting that this

mutant protein possesses catalytic activity and is con-centrated in the Golgi apparatus on its way to being discharged In keeping with this morphological obser-vation we found a low but significant enzyme activity

in both the homogenate and culture medium of the cells expressing TNSALP (1559delT) (Fig 7A) How-ever, an immunoblotting experiment demonstrated that the amount of TNSALP (1559delT) in the cell was less than one tenth of that of the wild type at steady state (Fig 7B, lanes 1 and 6), probably reflecting its rapid degradation as shown in Fig 5 Taking these values into consideration, the relative specific enzyme activity

of the mutant protein was calculated to be about one third of that of the wild type (Fig 7C) In contrast to the culture medium of the cells expressing the wild type enzyme, very high enzyme activity was detected in that of the cells expressing sTNSALP (Fig 7A), con-sistent with a metabolic labeling study showing that sTNSALP is rapidly secreted out of the cell (Fig 4C)

Aggregation of TNSALP (1559delT) Previously, we have reported that several TNSALP missense mutants tend to form a disulfide-bonded high-molecular-mass aggregate in transfected cells pre-sumably due to defective folding and random associ-ation of mutant proteins [10–14] To investigate if this

is also the case for TNSALP (1559delT), the newly synthesized mutant protein was immunoprecipi-tated and analysed by SDS⁄ PAGE under reducing or nonreducing condition TNSALP (1559delT) formed a large aggregate bonded by multiple disulfide-bonds at the top of the resolving gel (Fig 8A, lanes 2 & 4) In contrast, only a small amount of the wild type enzyme formed the aggregate (lanes 1 and 3) The aggregate

saponin - saponin +

TNSALP

1559delT

Fig 6 Cytohistochemical staining for

alka-line phosphatase Cells expressing TNSALP

or TNSALP (1559delT) were stained for

alka-line phosphate activity in the absence or

presence of saponin.

thus found in the cells expressing the wild type could

be GPI-anchor-less molecules, which are retained in

the ER (Fig 3) Note that the secreted

TNS-ALP (1559delT) also formed large aggregates (Fig 8A,

lanes 6 & 8) Addition of dithiothreitol in the culture

medium did not enhance the secretion of the mutant,

but rather inhibited it (results not shown) In good

agreement with the SDS⁄ PAGE, sucrose gradient

cen-trifugation further demonstrated that TNSALP

(1559delT) tends to form large aggregates

Consider-able amount of cellular activity and most of secreted

activity was recovered in the bottom three fractions

(Fig 8B) In contrast, sTNSALP peaked at fraction 7

(Fig 8B) Because the wild type enzyme also appeared

in fractions 6 and 7 in a similar analysis [12,13], this

result indicates that sTNSALP forms a dimer

Import-antly, Km values estimated by Lineweaver–Burk plots

were 4.3· 10)4m (wild type, cell homogenate),

1.9· 10)4m [TNSALP (1559delT), fractions 10–12 of

the medium] and 5.5· 10)4m (sTNSALP, medium)

This finding indicates that the C-terminal extension of

TNSALP (1559delT) does not significantly affect the substrate affinity of this mutant, thus differentiating TNSALP (1559delT) from other missense TNSALP mutants possessing no catalytic activity, such as TNS-ALP (R54C), TNSTNS-ALP (N153D), TNSTNS-ALP (E218G), TNSALP (D289V) and TNSALP (G317D) [10–14] Addition of dithiothreitol into the culture media and cell lysates of the cells expressing TNSALP (1559delT) did not enhance the enzyme activity (results not shown)

Replacement of three cysteines with serine residues in the C-terminus region

Despite its aggregation state, TNSALP (1559delT) shows catalytic activity comparable to that of the wild type and sTNSALP as described above We therefore speculated that TNSALP (1559delT) becomes correctly folded and assembled by the time that the cysteine resi-dues in the C-terminal region emerge through the tran-slocon of the ER, and that it eventually undergoes

1 2.5 5 10 10 10 10

1 2 3 4 5 6 7

80 kDa

66 kDa

TNSALP sTNSALP 1559delT

0

500

1000

1500

2000

2500

3000

TNSALP sTNSALP 1559delT

0 500 1000 1500 2000 2500 3000

B

Fig 7 Enzyme activity of TNSALP (1559delT) (A) COS-1 cells, which had been transfected with the plasmids encoding the wild type TNSALP, sTNSALP or TNSALP (1559delT), were cultured for 24 h and then homogenized in the 50 m M Tris ⁄ HCl (pH 7.5) The cell homogenates (white bars) and media (black bars) were assayed for alkaline phosphatase and expressed in unit per mg protein (cell) or unit per mL culture medium, respectively (B) In addition to the cell homogenates (lanes 1–6) prepared as described in (A), cells expressing TNSALP (1559delT) were incubated in the presence of of LLnL (10 l M ) for 24 h and then homogenized (lane 7) The homo-genates were analysed by immunoblotting with anti-TNSALP The numbers above the fluorogram shows the amounts (lg) of pro-tein applied on SDS ⁄ PAGE (C) The relative specific enzyme activities of the cell homo-genates prepared from cells expressing TNSALP or TNSALP (1559delT) described as

in A were calculated based on the relative amount (10 : 1) of both proteins in the homogenates as described in B (ordinate, arbitrary unit).

multiple cross-linking reactions via the cysteine

resi-dues To address this possibility, three cysteines were

substituted for serine residues in the C-terminal

ex-tension of TNSALP (1559delT) (Fig 1) Initially we

attempted to simultaneously replace all three cysteine

residues at positions 506, 521 and 577 However, only

two plasmids were obtained in which two out of three

cysteine residues were replaced [TNSALP

(1559delT-C506C⁄ C521S ⁄ C577S), TNSALP (1559delT-C506S⁄

C521C⁄ C577S)] When these two proteins were expressed in COS-1 cells, the amount of the large aggregate was markedly reduced and instead the cross-linked dimer became prominent (Fig 9A, lanes 11–14) Note the decrease in the aggregate on the stacking gel Next, we introduced the third mutation into TNSALP (1559delT-C506S⁄ C521C ⁄ C577S) The aggre-gation state was dramatically changed in the cells expressing TNSALP (1559delT-C506S⁄ C521S ⁄ C577S)

1 2 3 4 5 6 7 8

red nonred red nonred cell medium

90 kDa

66 kDa

80 kDa

0 5 10 15 20 25

1 2 3 4 5 6 7 8 9 10 11 12 0

1 2 3 4 5

1 2 3 4 5 6 7 8 9 10 11 12

0 500 1000 1500 2000 2500 3000 3500 4000 4500

1 2 3 4 5 6 7 8 9 10 11 12 0

10 20 30 40 50 60 70 80 90

1 2 3 4 5 6 7 8 9 10 11 12

1559delT (medium) 1559delT (cell)

c c

A

B

Fig 8 Sucrose-density-gradient analysis of

TNSALP (1559delT) (A) Cells expressing

TNSALP (lanes 1, 3, 5 and 7) or TNSALP

(1559delT) (lanes 2, 4, 6 and 8) were

continuously labeled with [ 35

S]methion-ine ⁄ cysteine for 4 h The media and cell

lysates were subjected to

immunoprecipita-tion The immune complexes were boiled in

the absence (nonreducing condition) or

pres-ence (reducing condition) of

2-mercaptoeth-anol and analysed by SDS ⁄ PAGE, followed

by fluorography An arrowhead indicates the

top position of the resolving gel Left lane:

14

C-methylated protein markers of 200,

97.4, 66 and 46 kDa (B) After 24 h

post-transfection, the lysates and media prepared

from cell cultures expressing sTNSALP or

TNSALP (1559delT) were directly applied on

the top of sucrose-density-gradient analysis

(5–35%) After centrifugation, each 400 lL

fraction was collected from the top of the

gradient and assayed for alkaline

phospha-tase activity (ordinate, unit per mL fraction).

BSA (b, 68 kDa), alcohol dehydrogenase

(a, 141 kDa) and catalase (250 kDa) were

loaded on to a separate gradient as

mole-cular mass markers.

Not only the aggregate but also the covalently linked

dimer almost disappeared (Fig 9A, lanes, 15 and 16)

This modified TNSALP (1559delT) was found to

sedi-ment at a dimer position as judged by sucrose-density

centrifugation (Fig 9B) We therefore concluded that

TNSALP (1559delT-C506S⁄ C521S ⁄ C577S) formed a

noncovalently assembled dimer similarly to sTNSALP

(Fig 8B) and the wild type enzyme [12,13] As

expected, TNSALP (1559delT-C506S⁄ C521S ⁄ C577S)

was secreted threefold more than TNSALP (1559delT)

(Fig 9B; compare ordinates)

Discussion

TNSALP (1559delT) is a large-sized secretory protein lacking GPI

A growing number of genetic diseases have been rela-ted to defective post-translational folding and resultant degradation in the ER as part of the ER quality con-trol system [22–24] TNSALP missense mutant pro-teins, in particular associated with severe form hypophosphatasia, fall into this category The missense

C M C M C M C M C M C M C M C M

red nonred

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

90 kDa

a

dimer

0 50 100 150 200 250 300 350

1 2 3 4 5 6 7 8 9 10 11 12

0 20 40 60 80 100 120

1 2 3 4 5 6 7 8 9 10 11 12

A

B

Fig 9 Replacement of the cysteine residues in the C-terminal extension (A) Cells were transfected with pALTER  -MAX encoding TNS-ALP (1559delT) (lanes 1, 2, 9 and 10), pALTER-MAX encoding TNSALP (1559delT-C506S ⁄ C521C ⁄ C577S) (lanes 3, 4, 11 and 12), pALTER 

-MAX encoding TNSALP (1559delT-C506C ⁄ C521S ⁄ C577S) (lanes 5, 6, 13 and14) or pAltermax encoding TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) (lanes 7, 8, 15 and 16) After 24 h the cells were continuously labeled with [ 35 S]methionine ⁄ cysteine After 3 h, the media (M) and cell lysates (C) were subjected to immnoprecipitation Iodoacetoamide was added to both the cell lysates and media (final concen-tration of 25 m M ) The immune complexes were analysed by SDS⁄ PAGE in the absence (nonreducing condition) or presence (reducing con-dition) of 2-mercaptoethanol, followed by fluorography Double and single arrowheads indicate the top of the stacking and resolving gels, respectively Left lane: 14 C-methylated protein markers of 200, 97.4, 66, 46 and 30 kDa (B) After 24 h post-transfection, the media were removed from the cell cultures expressing either TNSALP (1559delT) or TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) [1559delT (serines)] and directly applied on the top of the sucrose-density-gradient After centrifugation, each 400 lL fraction was collected from the top of the gradi-ent and assayed for alkaline phosphatase activity (ordinate, unit per mL fraction) BSA (b, 68 kDa), alcohol dehydrogenase (a, 141 kDa) and catalase (c, 250 kDa) were loaded on to a separate gradient as molecular mass markers.

mutations such as TNSALP (R54C), TNSALP

(N153D), TNSALP (E218G), TNSALP (D289V) and

TNSALP (G317D) are causes for severe molecular

phenotypes and exhibit only negligible alkaline

phos-phatase activity when expressed in the cell ectopically

These mutants were found to form disulfide-bonded

high-molecular-mass aggregates and accumulate in the

ER and⁄ or cis-Golgi, followed by degradation via the

proteasome In contrast to these missense mutants,

TNSALP (1559delT) is unique in that it has the long

C-terminal extension due to the frameshift mutation

In vitro translation⁄ translocation experiments

demon-strated that the translational product (66 kDa) of the

mutant protein is larger than that of the wild type by

 12 kDa, compatible with an additional 80 amino

acid residues at C-terminus (Fig 1) This 66 kDa

prod-uct becomes the 80 kDa form in the presence of the

microsome Probably the increase in molecular mass is

solely due to the acquisition of N-linked

oligosaccha-rides, as supported by two lines of evidence First, the

molecular shift was remarkably diminished when

trans-lation⁄ translocation experiments were carried out in

the presence of an inhibitor of N-linked

oligosaccha-ride attachment Second, the 80 kDa form was

conver-ted into the 66 kDa form by digestion with PNGase F

Consistent with the in vitro translation, we observed

the 80 kDa form immediately following a pulse-period

in the cultured cells expressing TNSALP (1559delT)

(Fig 4A, lane 4)

Another feature of this mutant is its solubility In

contrast with the missense mutants mentioned above,

TNSALP (1559delT) is a soluble enzyme lacking a

GPI-anchor This was examined by phase separation

using Triton X-114 The wild type enzyme is largely

partitioned into the detergent phase and moved into

the aqueous phase only after PI-PLC digestion

(Fig 3) TNSALP (1559delT) was exclusively

parti-tioned into the aqueous phase without PI-PLC

diges-tion As a control, a soluble truncated form of

TNSALP (sTNSALP) was also partitioned into the

aqueous phase, further supporting the hypothesis that

TNSALP (1559delT) lacks GPI

With respect to secretion, it is of interest that several

missense TNSALP mutant proteins are reported to be

secreted out of the cell, such as sTNSALP, when they

are synthesized as soluble forms lacking GPI [25]

TNSALP (1559delT) forms an aggregate

and is degraded

Although TNSALP (1559delT) is a soluble enzyme, its

secretion was far less efficient than that of sTNSALP

(Fig 4A,C) Nevertheless, a small portion of TNSALP

(1559delT) progressed to the Golgi apparatus, acquired Endo H-resistance and was then released as the

90 kDa form into the medium (Fig 4B) Analyses by SDS⁄ PAGE and sucrose-density-gradient analysis demonstrated that TNSALP (1559delT) formed a disulfide-bonded high-molecular-mass aggregate in the transfected cells (Fig 8), implying that this aggregation state is probably a cause of impaired secretion of TNS-ALP (1559delT) The aggregation may lower the prob-ability of TNSALP (1559delT) being segregated into COP II vesicles at the exit site from ER and therefore the mutant protein remains longer in the lumen of the

ER and finally is diverted to the degradation pathway Because the degradation is blocked by inhibitors of proteasome function (Fig 5A,B), it is likely that TNS-ALP (1559delT) is eventually degraded in the

TNSALP (1559delT) is polyubiquitinated before being destroyed in the proteasome (Fig 5C) TNSALP (1559delT) is not the only TNSALP mutant protein that is degraded via the ubiquitin⁄ proteasome path-way TNSALP (D289V), which is associated perinatal hypophosphatasia, is another example [14] These find-ings suggest that the biosynthesis of TNSALP is under scrutiny of the ER quality control system Improperly folded and incorrectly assembled molecules are moved into cytoplasm in the early stage of the secretory path-way [26–28] However, much remains to be learned regarding the molecular mechanism leading to degra-dation from the ER How are mutant forms of TNSALP but not the wild type recognized and retrotranslocated into the cytoplasm? What type of ubiquitin ligase(s) is involved in the ubiquitination of TNSALP mutant proteins prior to destruction in the proteasome? Furthermore, as both TNSALP (1559delT) and TNSALP (D289V) are present in aggregate state, is it an obligatory process to reduce the disulfide-bonded aggregate prior to translocation

in an opposite direction? Two molecules have recently emerged as key components of the ER quality control system, namely a Man8GlcNAc2-binding lectin (EDEM) [29,30], and SCFFbs2 ubiquitin ligase com-plex, which specifically targets N-linked high-mannose-type oligosaccharide chains of glycoproteins [31] The involvement of EDEM and⁄ or SCFFbs2in the degrada-tion of TNSALP mutant proteins is currently being investigated

The aggregate form of TNSALP (1559delT) possesses enzyme activity

TNSALP (1559delT) retains the catalytic function comparable to the wild type enzyme, even though it