Characterization of 16- to 20-Kilodalton (kDa) Connective Tissue Growth Factors (CTGFs) and Demonstration of Proteolytic Activity for 38-kDa CTGF in Pig Uterine Luminal Flushings1

Abstract

Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor ss. Although the primary translational product of the pig CTGF gene is predicted to be of approximate Mr 38 000, pig uterine luminal flushings (ULF) contained 10- to 20-kDa CTGF proteins that were heparin-binding and mitogenic, whereas 38-kDa CTGF was not apparent. The N-termini of two microheterogeneous forms of 16-kDa CTGF, as well as 18-kDa and 20-kDa forms of CTGF, commenced at, respectively, Cys199, Ala197, Asp186, and Asp186 and did not correspond to intron-exon boundaries in the CTGF gene. Northern blotting revealed a single porcine (p) CTGF transcript of 2.4 kilobases in endometrium from Day 10 to 16 cycling or pregnant pigs. Ten- to twenty-kilodalton pCTGF proteins in ULF were stable for 48 h at 37 degreesC whereas native 38-kDa pCTGF was degraded within 10 min under the same conditions. CTGF-degrading activity in pig ULF was heat-sensitive and concentration- and time-dependent. Ten- to twenty-kilodalton CTGF levels in ULF peaked on Day 16 of the cycle and on Day 12 of pregnancy and were highly correlated with the levels of proteolytic activity for 38-kDa CTGF. Collectively these data suggest that bioactive 10- to 20-kDa CTGF proteins are generated in utero through limited proteolysis of the 38-kDa CTGF primary translational product.

Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1–matrix metalloproteinase substrates

“…Hashimoto et al (41) also recently described CTGF cleavage by soluble MMPs 1, 3, 7, and 13 at Met-194-Ile-195 within the CTGF linker, with a minor cleavage site at Ala-181-Tyr-182. CTGF is unstable in vivo, being converted into low molecular mass forms (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) by an unidentified protease (42). Interestingly, conditioned medium from serum-stimulated mouse fibroblasts did not degrade CTGF (43), suggesting that a cell surface protease was responsible for CTGF processing.…”

Kallikrein-related Peptidase 12 Hydrolyzes Matricellular Proteins of the CCN Family and Modifies Interactions of CCN1 and CCN5 with Growth Factors

“…Various forms of CCN2 were detected in pig uterine flushings resulting from in utero proteolytic digestion of the full-length protein (41)(42)(43). In rats, an N-terminally truncated CCN3 form predominates in brain tissue and in cerebrospinal fluid (44).…”

Connective Tissue Growth Factor CCN2 Interacts with and Activates the Tyrosine Kinase Receptor TrkA

“…CTGF is also synthesized and secreted as a 36-to 38-kD peptide, but different forms between 10 and 20 kD are also detected and are believed to be proteolytic products of the 38-kD form (53). The C-terminal 10-to 12-kD peptide is biologically active.…”