Serglycin Participates In Retention of α-Defensin In Granules During Myelopoiesis.

The mechanism(s) that target(s) proteins to granules in neutrophils is unknown. The intracellular proteoglycan serglycin has been shown to have important functions related to formation of several types of storage granules. We investigated the possible role of serglycin in the localization of the α-defensin, human neutrophil peptide 1 (HNP-1), the main azurophil granule protein in human neutrophils, using a murine model. Since mice do not express HNP-1 in neutrophils, we transfected the murine myeloblast cell line 32D and the murine promyelocyte cell line MPRO with HNP-1. Furthermore the transgene HNP-1 mouse was crossed with serglycin knock out (k.o.) mouse and the elastase k.o. mouse to investigate HNP-1 sorting and processing in wild type transgene mice and in transgene mice lacking serglycin or elastase.

32D and MPRO cells were transfected with a vector expressing proHNP-1 and blasticidin resistance gene. Taqman Assay (Applied Biosystems) specific for DEFA1, the gene for HNP-1, was used for Real Time PCR. Western blotting was performed according to standard procedures using rabbit anti-mouse HNP-1 proHNP antibodies. Sandwich ELISA was performed with affinity-purified anti-serglycin Ig as catching antibody and biotinylated rabbit anti-mouse HNP-1 or proHNP-as detecting antibody. The transgene HNP-1 mouse was crossed with the serglycin k.o. mouse and with the neutrophil elastase k.o. mouse. Pulse-chase biosynthesis of HNP-1 was performed on isolated murine bone marrow cells. Cells were pulsed for 1h and chased for 3h. Lysed cells and medium were immunoprecipitated with Sepharose coupled antibodies against proHNP-1 and HNP-1. Proteins were subjected to SDS-PAGE and stained with Coomassie blue. The SDS-gel was submerged in Amplify, dried, and placed in a Fuji BAS cassette, exposed overnight and examined on a Phosphorimager.

Both 32D cells and MPRO cells were transfected with a vector expressing proHNP-1. Expression of proHNP-1 was verified by Real Time PCR and Western blotting. Both 32D cells and MPRO cells were capable of processing proHNP-1 to mature HNP-1 as confirmed by mass spectrometry of affinity purified low mw immunoreactive peptide isolated from the transfected 32D cell line. In order to assess whether serglycin does in fact associate with HNP-1, we performed a sandwich ELISA on 32D cells transfected with proHNP-1. Cell lysate was applied to ELISA plates that had been absorbed with antibody against serglycin. The bound lysates were then probed with antibody against either proHNP-1 or mature HNP-1. HNP-1 was detected by antibody against mature HNP-1, but not with antibody against the prosegment of HNP-1, indicating that fully processed HNP-1, but not unprocessed HNP-1, associates with serglycin in 32D cells. HNP-1 was expressed in bone marrow cells from HNP-1 mice. Most HNP-1 was routed extracellularly in an unprocessed form as previously observed in human bone marrow, but significant processing occurred in both wild type and serglycin-/- mice. Fully processed HNP-1 was found in the medium from serglycin -/- mice to a much larger extent than in wild type mice indicating that serglycin is necessary for retaining fully processed HNP-1 in granules. As serglycin has been shown to be essential for expression of elastase in myloid cells in mice, the fact that HNP1 expressing, serglycin deficient mice seem capable of efficiently processing proHNP1, indicates that elastase is not a major protease involved in processing of proHNP1 in vivo. To address this more directly, we crossed the elastase k.o. mouse into the HNP1 expressing transgene. We were not able to detect any difference in the capacity for processing of HNP1 using pulse-chase biosynthesis.

In conclusion our results show that murine cells are capable of processing proHNP-1 and that serglycin associates with HNP-1. Processing does not seem to depend on neutrophil elastase. Biosynthesis of transgene HNP-1 mice demonstrates the need for serglycin to retain fully processed HNP-1 intracellularly.

No relevant conflicts of interest to declare.