Romano P R, Wang J, O'Keefe R J, Puzas J E, Rosier R N, Reynolds P R
Department of Orthopaedics, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, USA.
J Cell Sci. 1998 Mar;111 ( Pt 6):803-13. doi: 10.1242/jcs.111.6.803.
We have previously identified and partially cloned Band 17, a gene expressed in growth plate chondrocytes transiting from proliferation to hypertrophy. We now rename this gene HiPER1, Histidine Phosphatase of the Endoplasmic Reticulum-1, based on the results reported here. HiPER1 encodes two proteins of 318 (HiPER1(318)) and 449 (HiPER1(449)) amino acids, which are 20-21% identical to a group of yeast acid phosphatases that are in the histidine phosphatase family. HiPER1(449) is significantly more abundant than HiPER1(318), correlating with the abundance of the alternatively spliced messages encoding HiPER449 and HiPER318. Anti-HiPER1 antibodies detect two proteins of 53 and 55 kDa in growth plate chondrocytes that are absent in articular chondrocytes. We confirm that the 53 and 55 kDa proteins are HiPER1(449) by heterologous expression of the HiPER1(449) coding sequence in chick embryo fibroblasts. The 53 and 55 kDa proteins are glycosylated forms of HiPER1(449), as N-glycosidase F digestion reduces these proteins to 48 kDa, the predicted size of HiPER1(449) without the N-terminal signal sequence. Immunocytochemistry demonstrates that HiPER1(449) is found in chondrocytes maturing from proliferation to hypertrophy, but is not detectable in resting zone, deep hypertrophic zone or articular chondrocytes, a distribution that is consistent with the message distribution. HiPER1(449) was predicted to localize to the lumen of endoplasmic reticulum by an N-terminal signal sequence and by the C-terminal sequence Ala-Asp-Glu-Leu, which closely matches the consensus signal for ER retention, Lys-Asp-Glu-Leu. We confirm this prediction by demonstrating colocalization of HiPER1(449) with the ER protein HSP47 using dual-label immunofluorescence. PTHrP, a peptide that prevents hypertrophy in chondrocytes, suppressed HiPER1 and HiPER1(449) expression in vitro, an observation that further supports a role for HiPER1 in chondrocyte maturation. The yeast phosphatase homology, localization to the endoplasmic reticulum and pattern of expression suggest that HiPER1 represents a previously unrecognized intracellular pathway, involved in differentiation of chondrocytes.
我们之前已经鉴定并部分克隆了17号条带基因，该基因在从增殖向肥大过渡的生长板软骨细胞中表达。基于此处报道的结果，我们现在将该基因重新命名为HiPER1，即内质网-1组氨酸磷酸酶。HiPER1编码两种蛋白质，分别含318个氨基酸（HiPER1(318)）和449个氨基酸（HiPER1(449)），它们与组氨酸磷酸酶家族中的一组酵母酸性磷酸酶有20%-21%的同源性。HiPER1(449)的丰度明显高于HiPER1(318)，这与编码HiPER449和HiPER318的可变剪接信使的丰度相关。抗HiPER1抗体在生长板软骨细胞中检测到53 kDa和55 kDa的两种蛋白质，而在关节软骨细胞中不存在。我们通过在鸡胚成纤维细胞中异源表达HiPER1(449)编码序列，证实53 kDa和55 kDa的蛋白质是HiPER1(449)。53 kDa和55 kDa的蛋白质是HiPER1(449)的糖基化形式，因为N-糖苷酶F消化将这些蛋白质减少到48 kDa，即没有N端信号序列的HiPER1(449)的预测大小。免疫细胞化学显示，HiPER1(449)存在于从增殖向肥大成熟的软骨细胞中，但在静止区、深部肥大区或关节软骨细胞中检测不到，这种分布与信使分布一致。通过N端信号序列和C端序列Ala-Asp-Glu-Leu预测HiPER1(449)定位于内质网腔，该序列与内质网保留的共有信号Lys-Asp-Glu-Leu非常匹配。我们通过使用双标记免疫荧光证明HiPER1(449)与内质网蛋白HSP47共定位，证实了这一预测。甲状旁腺激素相关蛋白（PTHrP）是一种可防止软骨细胞肥大的肽，它在体外抑制HiPER1和HiPER1(449)的表达，这一观察结果进一步支持了HiPER1在软骨细胞成熟中的作用。酵母磷酸酶同源性、内质网定位和表达模式表明，HiPER1代表了一条以前未被认识的细胞内途径，参与软骨细胞的分化。
