In vivo processing of CXCL12α/SDF‐1α after intravenous and subcutaneous administration to mice

Abstract

CXCL12α has been shown to be selectively processed at the N- and C-termini in blood and plasma in vitro. In order to study the processing in vivo, several versions of CXCL12α were expressed and purified. The protein was administered either iv or sc to mice, and at different time points postadministration plasma was collected and analyzed. To detect modifications of the CXCL12α molecule in crude plasma a SELDI TOF-MS-based method was developed. Anti-CXCL12 antibodies were immobilized on the SELDI chip and CXCL12α binding to the antibodies was detected by SELDI-TOF-MS. The protein was found to be processed both at the C- and N-termini. The same processed CXCL12α forms as detected in vitro were found; however, in addition further processing was detected at the N-terminus, where altogether seven amino acids were removed. At the C-terminus the lysine was removed as has been seen in vitro, and no further processing was detected. The full-length CXCL12α disappeared within minutes after administration, whereas the processed forms of the protein were detectable for up to 6-8 h postadministration. The same processed forms appeared after iv and sc administration, only the kinetics was different. When the shortest processed form detected in plasma, 7ΔN1ΔC-CXCL12α, was administered directly, no further processed forms were detected. Interestingly, a version of CXCL12α containing a N-terminal methionine was protected against N-terminal processing in plasma in vitro; however, in vivo no protection was seen, the protein was processed in the same way as full-length CXCL12α.

Identification and Characterization of Circulating Variants of CXCL12 from Human Plasma: Effects on Chemotaxis and Mobilization of Hematopoietic Stem and Progenitor Cells

“…In the study of Antonsson et al mentioned earlier, all of the CXCL12 variants identified in our study were also found with the exception of CXCL12 [19]. The absence of CXCL12 might be related to species-specific differences in the neutrophil serine protease activities [42,43].…”

“…Importantly, these MMPs produce CXCL12 [34], a CXCL12 variant that was also identified in a study by Antonsson subsequent to an in vivo application of CXCL12 in mice [19]. In contrast, CXCL12 was not identified in our in vitro CXCL12-processing experiments performed with human granulocytes and was not identified in the purified material from blood filtrate, although the CXCL12[26-88]-producing protease MMP-9 is primarily expressed in granulocytes.…”

“…They showed that s.c. or i.v. adminstration of CXCL12 revealed no additional proteolytic product but instead showed slow elimination of CXCL12 ] within 6-8 h postadministration [19].…”

“…plasma that is accompanied by a mobilization of HPCs [14][15][16][17]. However, it is not known whether CXCL12 in blood plasma is immediately inactivated by proteolytic degradation, or whether the liberated CXCL12 stays functional for a significant time period, given that proteolytic inactivation of CXCL12 by DPPIV in blood plasma takes place with a half life of less than 1 min and that physiological CXCL12 concentrations in blood plasma are proteolytically degraded within 1-2 min [18,19].…”

“…CXCL12 is a proteolytic product of the metalloprotease DPPIV, which has been suggested to degrade CXCL12 in the circulation with high efficacy [18,19]. CXCL12 and CXCL12 are proteolytic products that are generated by neutrophils [5].…”