Okagaki T, Weber F E, Fischman D A, Vaughan K T, Mikawa T, Reinach F C
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
J Cell Biol. 1993 Nov;123(3):619-26. doi: 10.1083/jcb.123.3.619.
A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.
来自多种物种的肌球蛋白结合蛋白的一个共同特征是存在数量可变的与免疫球蛋白C2或纤连蛋白III型重复序列同源的相关内部基序。尽管人们对这些基序的潜在功能很感兴趣，但尚无研究小组能明确证明这些序列在肌肉细胞内或细胞外的功能。我们已完成鸡骨骼肌中MyBP-C（C蛋白）快速型同工型的核苷酸序列测定。推导的氨基酸序列显示MyBP-C中有七个免疫球蛋白C2结构域和三个纤连蛋白III型基序。对纯化的MyBP-C进行α-胰凝乳蛋白酶消化产生四个肽段。对这些肽段进行氨基末端测序使我们能够确定每个肽段在蛋白质一级结构上的位置。28-kD肽段包含MyBP-C的氨基末端序列，包括第一个C2重复序列。随后是两个内部肽段，一个5-kD的肽段仅包含第一个和第二个C2基序之间的间隔序列，以及一个95-kD的片段，包含五个C2结构域和三个纤连蛋白III型基序。MyBP-C的羧基末端序列存在于一个14-kD的肽段中，该肽段仅包含最后一个C2重复序列。我们研究了这些片段与重组（合成）肌球蛋白丝的结合特性。只有羧基末端的14-kD肽段能够以高亲和力结合肌球蛋白。氨基末端的28-kD片段没有肌球蛋白结合能力，而长的内部100-kD肽段与肌球蛋白的结合非常弱。我们在大肠杆菌中表达并纯化了14-kD肽段。重组蛋白与肌球蛋白表现出饱和结合，其亲和力与通过蛋白酶消化获得的14-kD片段相当（在约0.5 microM时达到最大结合的1/2）。这些结果表明，与肌球蛋白丝的结合主要局限于MyBP-C的最后102个氨基酸。分子的其余部分（1032个氨基酸）可能与肌联蛋白、MyBP-H（H蛋白）或细肌丝成分相互作用。对迄今为止已测序的五种MyBP羧基末端存在的高度保守的免疫球蛋白C2结构域（人慢速和快速MyBP-C、人和鸡MyBP-H以及鸡MyBP-C）进行比较，以鉴定这些与肌球蛋白结合的免疫球蛋白C2重复序列特有的残基。
