Yoko Ishida

Zinc Transport Complexes Contribute to the Homeostatic Maintenance of Secretory Pathway Function in Vertebrate Cells

Zinc transporters play important roles in a wide range of biochemical processes. Here we report an important function of ZnT5/ZnT6 hetero-oligomeric complexes in the secretory pathway. The activity of human tissue-nonspecific alkaline phosphatase (ALP) expressed in ZnT5–ZnT7–/– cells was significantly reduced compared with that expressed in wild-type cells as in the case of endogenous chicken tissue-nonspecific ALP activity. The inactive human tissue-nonspecific ALP in ZnT5–ZnT7–/– cells was degraded by proteasome-mediated degradation without being trafficked to the plasma membrane. ZnT5–ZnT7–/– cells showed exacerbation of the unfolded protein response as did the wild-type cells cultured under a zinc-deficient condition, revealing that both complexes play a role in homeostatic maintenance of secretory pathway function. Furthermore, we showed that expression of ZnT5 mRNA was up-regulated by the endoplasmic reticulum stress in various cell lines. The up-regulation of the hZnT5 transcript was mediated by transcription factor XBP1 through the TGACGTGG sequence in the hZnT5 promoter, and this sequence was highly conserved in the ZnT5 genes of mouse and chicken. These results suggest that zinc transport into the secretory pathway is strictly regulated for the homeostatic maintenance of secretory pathway function in vertebrate cells.

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

In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations 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 TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein 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 aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C‐terminal 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).

Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406. Structural importance of the crown domain.

Hypophosphatasia, a congenital metabolic disease related to the tissue‐nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS‐1 cells and in stable CHO‐K1 Tet‐On cells. Compared with the wild‐type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild‐type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol‐anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild‐type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.

Aberrant interchain disulfide bridge of tissue-nonspecific alkaline phosphatase with an Arg433 → Cys substitution associated with severe hypophosphatasia

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 hypophosphatasia, 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 centrifugation 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 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 TNSALP(R433C).

Molecular characterization of tissue-nonspecific alkaline phosphatase with an Ala to Thr substitution at position 116 associated with dominantly inherited hypophosphatasia.

Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia, an inborn error of bone and teeth metabolism associated with reduced levels of serum alkaline phosphatase activity. A missense mutation (c.346G>A) of TNSALP gene, which converts Ala to Thr at position 116 (according to standardized nomenclature), was reported in dominantly transmitted hypophosphatasia patients (A.S. Lia-Baldini et al. Hum Genet. 109 (2001) 99-108). To investigate molecular phenotype of TNSALP (A116T), we expressed it in the COS-1 cells or Tet-On CHO K1 cells. TNSALP (A116T) displayed not only negligible alkaline phosphatase activity, but also a weak dominant negative effect when co-expressed with the wild-type enzyme. In contrast to TNSALP (W, wild-type), which was present mostly as a non-covalently assembled homodimeric form, TNSALP (A116T) was found to exist as a monomer and heterogeneously associated aggregates covalently linked via disulfide bonds. Interestingly, both the monomer and aggregate forms of TNSALP (A116T) gained access to the cell surface and were anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Co-expression of secretory forms of TNSALP (W) and TNSALP (A116T), which are engineered to replace the C-terminal GPI anchor with a tag sequence (his-tag or flag-tag), resulted in the release of heteromeric complexes consisting of TNSALP (W)-his and TNSALP (A116T)-flag. Taken together, these findings strongly suggest that TNSALP (A116T) fails to fold properly and forms disulfide-bonded aggregates, though it is indeed capable of interacting with the wild-type and reaching the cell surface, therefore explaining its dominant transmission.

Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia

Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-β-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to gain access to the cell surface.

Preliminary study of food space and head movement during food intake in skeletal Class III malocclusion cases

食物摂取時の開口量は, 常に食物の大きさよりも大きく, その差すなわち食品空隙はほぼ一定の値を取ることが知られている.また, この開口運動には頭部運動が随伴しており, 円滑な開閉口運動に寄与している.つまり開口運動は, 下顎運動と頭部運動が複合した運動であるといえる.ところで, 個々の骨格, 咬合の違いや下顎運動の特徴などによって至適な開口量や食品空隙量もまた異なる可能性が考えられ, 骨格性下顎前突症例における食物摂取時の食品空隙および頭部運動の特徴について, 興味のもたれるところである.そこで, 本研究では, 骨格性下顎前突症例の食物摂取時開口運動における下顎運動と頭部運動を観察し, 正常咬合者との比較を試みた.被験者は, 骨格性下顎前突症例 (女性3名, 男性1名, 計4名) , 正常咬合者 (女性3名, 男性1名, 計4名) とした.6自由度顎運動測定装置を用い, 食物摂取時の開口量および食品空隙量, 頭部運動量, 下顎頭移動量の測定を行い, 以下の結果を得た.1.骨格性下顎前突症例群においても正常咬合者群と同様に食品空隙が存在し, その量は食品の大きさの増加に伴って減少する傾向を示した.下顎前突症例群と正常咬合者群との間では, すべての被験食品の大きさにおいて食品空隙量に有意な差は認められなかった.2.骨格性下顎前突症例群においても, 開口運動に付随して頭部運動が観察されたが, 頭部運動量が正常咬合者群に類似した値を示す被験者と, 正常咬合者群よりも大きな値を示す被験者が存在した.今後さらに被験者を増やし, 精査する所存である.