Processing of α-Defensin in Subcellular Fractionations of Promyelocytic Cell Line PLB-985
Authors: Glenthøj Andreas , Cowland Jack B , Borregaard Niels 
Published: October 2019

Abstract 2163

Introduction:

α-defensins – in neutrophils also known as human neutrophil peptides (HNPs) – are the dominating proteins of neutrophil azurophil granules. They are synthesized in promyelocytes and myelocytes as proHNP, but only processed to mature HNP in promyelocytes. Yet, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unsettled. Thus neither the processing enzyme of proHNP nor the localization of processing has been identified. We studied processing of proHNP in subcellular fractions of the human promyelocytic cell line PLB-985 and identified fractions in which processing occurs. Furthermore, different proteinase inhibitors were employed to test their ability to inhibit PLB-985 proteases from processing proHNP.

Methods:

Subcellular fractionation of PLB-985 cells was performed by nitrogen cavitation and sedimentation of the postnuclear supernatant on a two-layer Percoll density gradient. Fractions of 1 ml each were collected from the bottom of the gradient using a fraction collector. Myeloperoxidase (MPO) and calnexin were measured by western blotting in all fractions and used as markers for azurophil granules and endoplasmic reticulum respectively. 35S-labelled proHNP was obtained by affinity chromatography on medium from a prodefensin expressing cells line incubated overnight in medium containing 35S-cysteine/methionine. 35S-labelled proHNP was incubated with subcellular fractions from PLB-985 cells, subjected to SDS-Tricine-PAGE, and visualized by fluorography. Inhibition of proHNP processing was probed with a battery of protease inhibitors: PMSF, aprotinin, leupeptin, EDTA, pepstatin A, chymostatin, and E-64. For biosynthesis studies, PLB-985 cells were incubated with in medium containing 35S-cysteine/methionine before subcellular fractionation, immunoprecipitation, SDS-Tricine-PAGE, and fluorography.

Results:

35S-labelled proHNP was incubated with subcellular fractions from PLB-985 cells and tested by SDS-Tricine-PAGE and fluorography. Most extensive processing occurred in fractions positive for both calnexin (marker for ER) and MPO, which is actively synthesized in the promyelocyte. The heavier granules fractions were only positive for MPO and showed a lesser degree of processing. To test where proHNP is processed in promyelocytes, PLB-985 cells were pulsed overnight in medium containing 35S-cysteine/methionine. After 20h cells, subcellular fractionation was performed and fractions immunoprecipitated with an anti-defensin antibody coupled to Sepharose beads. Immunoprecipitate was subjected to SDS-Tricine-PAGE and fluorography, which showed proHNP in fractions with both calnexin and MPO, but not in heavier fractions representing granules. Processed HNP was present in all proHNP-containing fractions as well as in the protein dense granule fractions. 35S-labelled proHNP was incubated with PLB-985 lysate and protease inhibitors to test, which protease classes in the promyelocyte are capable of proHNP processing. Of the inhibitors tested, only the serine protease inhibitor PMSF and to a minor degree the aspartyl peptidase inhibitor pepstatin A showed significant inhibition of proHNP processing.

Conclusion:

We studied posttranslational processing of proHNP using a human promyelocytic cell line model. proHNP processing proteases were identified in pre-granular structures of the biosynthetic pathway as well as in granule fractions. Radiolabeling showed processed HNP early in the biosynthetic pathway as well as in granules, but proHNP was only detected in pre-granule fractions consistent with proHNP being processed before localization to granules. This is in accordance with recent findings indicating that processed HNP binds to the proteoglycan serglycin, which is primarily localized in the Golgi apparatus. Our findings showed the serine protease inhibitor PMSF to be a strong inhibitor of proHNP processing in the promyelocyte. This is in accordance with previous findings of the three major proteases of the promyelocyte – the serine proteases neutrophil elastase, cathepsin G, and proteinase 3 – being able to process proHNP in vitro.

Disclosures:

No relevant conflicts of interest to declare.