Primary structure and tissue distribution of FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal morphogenesis
Primary structure and tissue distribution of FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal morphogenesis
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DOI：
10.1074/jbc.271.42.26131
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发表时间：
1996-10-18
影响因子：
4.8
通讯作者：
Luyten, FP
中科院分区：
文献类型：
--
作者：
Hoang, B;Moos, M;Luyten, FP
制备关节软骨提取物以表征具有体内软骨形成活性的蛋白质级分（Chang，S.，Hoang，B.，托马斯，JT，Vukicevic，S.，Luyten，F. P.，Ryba，N. J.P.，科扎克角一、Reddi，A. H、和Woos，M.（1994）J.Biol.Chem.269，28227-28234）。胰蛋白酶消化高度纯化的软骨形成蛋白组分允许通过氨基酸测序鉴定几种独特的肽。我们发现了一个新的cDNA编码推导的36 kDa的蛋白质，通过使用来自一个30个残基的肽在逆转录聚合酶链反应的简并寡核苷酸引物。其N-末端结构域显示出与果蝇基因frizzled的相应区域相似的50%的氨基酸同一性，该基因在发育过程中与毛发极性的特化有关。开放阅读框的亲水性和结构分析揭示了信号肽和疏水结构域的存在，随后是多个潜在的丝氨酸/苏氨酸磷酸化位点和富含丝氨酸的C末端。原代牛关节软骨细胞和转染COS细胞的细胞分级分离研究表明，该蛋白是膜相关的。人胚胎切片的原位杂交和免疫染色显示，主要表达周围的骨化骨原基，随后在骨骺的软骨细胞中的梯度分布，减少对主要骨化中心。转录本存在于颅面结构中，但不存在于椎体中。因为它主要在胚胎和胎儿发育（6-13周）期间发育中长骨的软骨核心中表达，并且与极性决定基因frizzled同源，我们认为该基因（我们命名为frzb）参与哺乳动物骨骼的形态发生。
Articular cartilage extracts were prepared to characterize protein fractions with in vivo chondrogenic activity (Chang, S., Hoang, B., Thomas, J. T., Vukicevic, S., Luyten, F. P., Ryba, N. J. P., Kozak, C. A., Reddi, A. H., and Woos, M. (1994) J. Biol. Chem. 269, 28227-28234). Trypsin digestion of highly purified chondrogenic protein fractions allowed the identification of several unique peptides by amino acid sequencing. We discovered a novel cDNA encoding a deduced 36-kDa protein by using degenerate oligonucleotide primers derived from a 30-residue peptide in reverse transcription polymerase chain reactions. Its N-terminal domain showed similar to 50%, amino acid identity to the corresponding region of the Drosophila gene frizzled, which has been implicated in the specification of hair polarity during development. Hydropathy and structural analyses of the open reading frame revealed the presence of a signal peptide and a hydrophobic domain followed by multiple potential serine/threonine phosphorylation sites and a serine-rich C terminus. Cell fractionation studies of primary bovine articular chondrocytes and transfected COS cells suggested that the protein is membrane-associated. In situ hybridization and immunostaining of human embryonic sections demonstrated predominant expression surrounding the chondrifying bone primordia and subsequently in the chondrocytes of the epiphyses in a graded distribution that decreased toward the primary ossification center. Transcripts were present in the craniofacial structures but not in the vertebral bodies. Because it is expressed primarily in the cartilaginous cores of developing long bones during embryonic and fetal development (6-13 weeks) and is homologous to the polarity-determining gene frizzled, we believe that this gene, which we named frzb, is involved in morphogenesis of the mammalian skeleton.