Kyomi Ibaraki

Murine bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic mapping.

Bone sialoprotein (BSP) is a small ( -70 ,000 Mr), highly post-translationally modified protein that is an abundant noncollagenous component of the bone matrix (Fisher et al. 1983). With a combination of immunohistochemistry and in situ hybridization, it has been shown that in the human and rat, BSP is produced by bone-forming cells and mature osteoblasts, recently entombed osteocytes, osteoclasts, and hypertrophic chondrocytes within the growth plate (Bianco et al. 1991; Chen et al. 1991). The only nonskeletal source that shows expression of BSP is the mononucleated trophoblast cells and their multinucleated syncytia in the developing placenta. Studies in the rat also showed that BSP is expressed in the odontoblasts of the developing mandibular incisor (Chen et al. 1992b). In an in vitro model of differentiating fetal bovine osteoblasts, BSP mRNA was increased 120-fold at a time point that corresponded to a dramatic formation of mineralizing nodules and trabecular-like structures (Ibaraki et al. 1992). This localization of BSP to sites of new mineral formation implicates BSP in this process. To determine the structure and describe the expression of BSP in the mouse, we have cloned and sequenced mouse BSP cDNA. In this study we report the nucleotide sequence of the murine cDNA, its deduced amino acid sequence, and we report the mapping of the mouse BSP gene (Ibsp) to Chromosome (Chr) 5. Mouse BSP cDNA was cloned from a 16-day mouse embryo cDNA library in a lambda SHlox vector (Novogen Cat #69641-1) with a 1.8-kb insert of rat cDNA (Oldberg et al. 1988a) as a probe under moderate hybridization conditions (Sambrook et al. 1989). One clone contained an insert size of 1.3 kb and was sequenced completely through-

MOLECULAR AND CELLULAR BIOLOGY OF THE MAJOR NONCOLLAGENOUS PROTEINS IN BONE

Publisher Summary This chapter discusses the molecular and cellular biology of the major non-collagenous proteins in bone. It also presents the structure and expression of a protein called osteonectin. By cDNA cloning and subsequent protein sequencing, it has become apparent that osteonectin is widely distributed in the body and likely to have multiple functions in mineralized and nonmineralized tissues as well. Despite its wide tissue distribution, it is, comparatively, highly expressed in bone. For this reason, the osteonectin gene has been used as a model to study transcriptional control in human- and bovine-derived cultured bone cells. The chapter also describes the two cell attachment bone matrix proteins, osteopontin and bone sialoprotein. While they both share the same small cell attachment motif, they are otherwise distinct in structure and in their extensive posttranslational modifications. The transcriptional control of the two genes also appears to be distinct and varies considerably with direct hormone/vitamin treatment as well as during development and with cellular transformation.

Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

Tetranectin is a plasminogen-binding tetrameric protein originally isolated from plasma. Expression of tetranectin appears ubiquitous, although particularly high expression is noted in the stroma of malignant tumors and during mineralization. To dissect the molecular basis of tetranectin gene regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76% identity and 87% similarity at the amino acid level. Sequence comparisons between mouse and human tetranectin and some C-type lectins confirmed a complete conservation in the position of six cysteines as well as numerous other amino acid residues, indicating an essential structure for potential function(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed in human. Although additional minor bands of 1.5 and 3.3 kb were found in Northern blots, RT-PCR (reverse transcription polymerase chain reaction) analysis failed to provide evidence that these minor bands are products of the tetranectin gene. Finally, the genetic map location for this gene, Tna, was determined to be on distal mouse Chromosome (Chr) 9 by analysis of two sets of multilocus crosses.