Molecular analysis of chicken embryo SPARC (osteonectin)

Abstract

SPARC is a secreted glycoprotein that modulates cell shape and cell-matrix interactions. Levels of SPARC are increased at sites of somitogenesis, osteogenesis, and angiogenesis in the embryo and during wound repair in the adult. We have cloned and characterized SPARC from chicken embryo. A 2.2-kbp cDNA, obtained by a novel use of the polymerase chain reaction, was determined to encode a 298-residue protein that is 85% identical to human SPARC. Antigenic sites in particular appear to be highly conserved, as antibodies against C-terminal sequences of murine and bovine SPARC reacted with a 41 -43 kDa protein in chicken embryo extracts. Chicken SPARC can be defined by four sequence signatures: (a) a conserved spacing of 11 cysteine residues in domain 11, (b) the pentapeptide KKGHK in domain 11, which is contained within a larger region of 31 identical residues, (c) a 100% conserved region of 10 residues in domain 111, and (d) a C-terminal, calciumbinding EF-hand motif. SPARC mRNAs in the 10-day-old chicken embryo are represented by three sizes of 1.8, 2.2 and 3.0 kb. The relative steady-state levels for the 2.2-kb mRNA were determined as aorta 3 skeletal muscle > calvarium > vertebra > anterior limb > kidney > heart > brain > skin and lung 9 liver. The relative abundance of the 1.8-kb and 2.2-kb mRNAs varied among tissues and indicated that differential processing of SPARC mRNAs might occur. All three RNA species were detected by a cDNA probe for the N-terminal part of the coding region. Thus, the three mRNA species appear to arise from differential 3' splicing and/or polyadenylation. Collective evidence demonstrates that SPARC has been well-conserved during vertebrate evolution, a finding that indicates a fundamental role for this protein in development.

Down-regulation of the extracellular matrix protein SPARC in vSrc- and vJun-transformed chick embryo fibroblasts contributes to tumor formation in vivo

“…The two closelymigrating bands are therefore likely to correspond to two isoforms of mature SPARC with distinct glycosylation levels. This explanation is consistent with the existence of potential N-linked glycosylation sites at Asn 67 and Lys 95 in the avian protein (Bassuk et al, 1993).…”

“…The cDNA sequence encoding the secreted form of the avian SPARC, noted rSPARC, has been recovered from the original plasmid rSPARC-pCR II (Bassuk et al, 1993) as a 928 base pair, EcoRI ± EcoRI fragment, and inserted at EcoRI into the pH plasmid to generated pH-rSPARC. pH is a pBluescript II SK + derivative (Stratagene) in which the SacI to SalI fragment from the original polylinker has been modi®ed (Baguet and Castellazzi; unpublished data); the new polylinker contains the following primer/promoter sequences, restriction sites, and polyA sequence in the order: RP ± BssHII ± T3 ± SP6 ± ClaI ± HindIII ± PstI ± SalI/ AccI ± XbaI ± BamHI ± SmaI ± SacI ± EcoRI ± ClaI-polyA 30 ± NotI ± XhoI ± ApaI ± KpnI ± T7 ± BssHII ± M13.…”

“…A pH-rSPARC derivative was constructed that contains the natural peptide signal sequence (Met 717 to Ala 71; see Figure 1 in Bassuk et al, 1993) in front of Ala+1 from the rSPARC sequence and designated pH-SPARC sig+. To do this, two double-stranded, synthetic oligonucleotides linked together by means of an arti®cial S®I site have been inserted into pH-rSPARC open at the restriction sites HindIII from the pH polylinker and BglII from the coding sequence (covering amino acids Glu+14 to Leu+16).…”

Transcription factor ATF3 partially transforms chick embryo fibroblasts by promoting growth factor-independent proliferation

“…For this purpose, we analysed genes whose mRNA levels were either down-or upregulated in v-Jun(-m1)-transformed CEFs and which are thought to contribute to oncogenic transformation. These genes encode: (i) the extracellular matrix components ®bronectin (Norton and Hynes, 1987), decorin (Li et al, 1992), thrombospondin 2 (Tucker, 1993), collagen XII (WaÈ lchli et al, 1994), clusterin (Michel et al, 1989), SPARC (Bassuk et al, 1993), a2 (I) collagen (Lehrach (a) CEFs chronically infected either with R or R-ATF3 were transiently transfected with the reporter constructs 56jun2-tata-luciferase, 56collTRE-tata-luciferase, or tata-luciferase. Data were from three independent experiments with dierent primary cultures.…”

SPARC Is Expressed by Ganglion Cells and Astrocytes in Bovine Retina

“…However, in the central nervous system there appears to be a constitutive, relatively robust expression of SPARC. In fact, studies have reported low levels of SPARC protein and mRNA in the embryonic nervous system (Nomura et al 1989;Bassuk et al 1993) and Immunoblotting analysis of SPARC protein in the bovine adult retina. Protein isolated from bovine adult retina were separated on polyacrylamide gels (4ր10%) in the presence of DTT and were transferred electrophoretically to a nitrocellulose membrane.…”