Association of activated properdin with complexes of properdin with C3

An ELISA using antibody to properdin (P), followed by antibody to C3 to detect complexes of P with C3 (P-C3), detected low levels of P-C3 complexes in human serum and plasma samples. Incubating serum for 1 h at 37 degrees C increased the amount of P-C3 and diminished factor B hemolytic activity without altering total alternative pathway activity or C3 activity in serum. When P and C3 in incubated serum were analyzed by size exclusion HPLC, complexes of P-C3 were detected at retention times corresponding to molecular mass measuring in excess of 2 x 10(6) Da. Activation of serum with zymosan or cobra venom factor greatly increased the level of P-C3 and decreased alternative pathway hemolytic activity. Chromatography of proteins eluted from serum-treated zymosan detected a peak of P at 9.7 x 10(5) Da and a peak of P-C3 at 1.5 x 10(6) Da. Functional assays for activated properdin also revealed a peak of activity at 1.5 x 10(6) Da, congruent with the peak of P-C3. Native properdin was detected at 3.9 x 10(5) Da. When native properdin was added to properdin-depleted serum and incubated for 1 h at 37 degrees C, activated properdin was detected at the same position in the chromatograph as were P-C3 complexes. We conclude that incubation of serum at 37 degrees C produces complexes of P with C3, that exposure of serum to alternative pathway activators increases the amount of P-C3, and that generation of P-C3 complexes is associated with the presence of activated P.     

Biosynthesis of properdin

Properdin (P) is synthesized by the human promyelocytic cell line, HL-60, after differentiation with DMSO. The secreted P was physiochemically indistinguishable from purified plasma P. It was polymerized and able to bind to C3IBb-Sepharose but not to C3i-Sepharose. No extracellular precursor was present. The intracellular form, detected between 1 and 4 h after labeling, was similar but had a slightly lower Mr. It also bound reversibly to C3iBb-Sepharose, and polymers could be demonstrated by cross-linking. Pulse-chase experiments suggested the existence of an earlier, but undetectable, intracellular precursor(s). This form could not be immunoprecipitated even when harsh solubilization conditions and/or antibodies against reduced and denatured P were utilized. Studies with endoglycosidases F and H and tunicamycin indicated that the detectable intracellular precursor contains high mannose N-linked carbohydrate that is processed to the complex form before secretion. The sugars are not required for polymerization, secretion, or functional activity, or responsible for the electrophoretic heterogeneity. Polymerization of P is therefore an early intracellular event, perhaps carefully controlled to prevent anomalous aggregation.     

Genetic and physical mapping around the properdin P gene.

A CA repeat has been found on the human X chromosome within 16 kb of the gene encoding properdin P factor (PFC) and has been shown to be a highly informative marker. Two more polymorphic CA repeats were found in a cosmid containing DXS228. The CA repeats, and other markers from proximal Xp, were mapped genetically in CEPH families and the likely order of markers was established as Xpter-(DXS7, MAO-A, DXS228)-(PFC, DXS426)-(TIMP, OATL1)-DXS255-Xcen. This places PFC in the region Xp11.3-Xp11.23, thus refining previous in situ hybridization data. Two yeast artificial chromosomes (YACs) (440 and 390 kb) contain both PFC and DXS426, and one of them (440 kb) also contains TIMP. This confirms the genetic order TIMP-(PFC, DXS426). PFC and TIMP are located on the same 100-kb SalI/PvuI fragment of the 440-kb YAC. Given the genetic orientation of TIMP and (PFC, DXS426), this YAC can now serve as a starting point for directional walking toward disease genes located in Xp11.3-Xp11.2 such as retinitis pigmentosa (RP2) and Wiskott-Aldrich syndrome.     

Localization of the properdin structural locus to Xp11.23-Xp21.1

Properdin is a serum protein belonging to the alternative pathway of complement activation whose absence is often associated with fatal bacterial infections. Properdin deficiency segregates with an X-linked recessive pattern and its position has been recently refined by genetic linkage analysis to the proximal part of the X-chromosome short arm near the OTC and DXS7 loci. We have hybridized an 0.8-kb genomic clone encoding part of the human properdin gene to a panel of somatic cell hybrids retaining different portions of the human X chromosome and thereby localized the probe to Xcen-Xp21.1. Furthermore, in situ hybridization of the same probe to replication banded metaphase chromosomes refined this localization to the region Xp11.23-Xp21.1 (with a peak grain distribution in the region equivalent to Xp11.4). As OTC and DXS7 map to Xp21.1 and Xp11.3, respectively, the data presented here strongly suggest that the X-linked deficiency syndrome is due to a defect in the locus encoding the structural properdin gene or in a physically close regulatory locus.