Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4)

Serine proteases in cytoplasmic granules of neutrophils (NSPs),
namely neutrophil elastase, cathepsin G (CG) and proteinase 3, have
been under intense investigation for several decades. They are
mainly known for their role in intracellular killing of pathogens
and are also increasingly recognized as key regulators of innate
immune responses. In 2009, I identified a fourth serine protease in
neutrophils that has been completely overlooked and neglected so
far. The aim of this thesis was an in-depth biochemical and
functional characterization of this novel serine protease 4 (NSP4)
of human neutrophils. Using monoclonal antibodies to NSP4, the
distribution of NSP4 in normal human tissues was studied. NSP4 was
observed only in neutrophils and neutrophil precursors of the bone
marrow. The content of NSP4 in neutrophil lysates was about 20-fold
lower compared to CG. Nevertheless, NSP4 was found to be released
into the supernatant upon neutrophil activation. NSP4 could be
further identified as a novel azurophil granule protein of
neutrophils by Western blot analyses of subcellular fractions. For
the functional analysis, the production and yield of recombinant
NSP4 was clearly improved using different expression systems and
DNA construct modifications. The proteolytic specificity was analyzed
using E. coli peptide libraries, mass spectrometry and several
synthetic peptide libraries. All these analyses clearly revealed an
arginine specificity for NSP4. Consistent with this, NSP4 was
strongly inhibited by heparin-accelerated antithrombin and C1
inhibitor and, with lower efficacy, by α1-proteinase inhibitor
(α1PI). The data allowed me to generate an NSP4-specific α1PI
variant that was shown to form covalent complexes with all NSP4 of
neutrophil lysates and supernatants of activated neutrophils. This
finding strongly indicated that NSP4 is fully processed and stored
as an already activated enzyme in azurophil granules. In addition,
dipeptidyl peptidase I (DPPI) was identified as the activator of
NSP4 in vivo, as DPPI deficiency resulted in complete absence of
NSP4 in a Papillon-Lefèvre patient. Analysis of cell-based calcium
assays revealed that proteinase-activated receptor-2 may represent
a potential natural substrate of NSP4. So far, NSP4-deficient mice
did not show an abnormal phenotype under clean housing conditions.
Activation of isolated neutrophils by phorbol esters or immune
complexes was also not impaired. This study establishes NSP4 as the
only arginine-specific pre-activated serine protease stored in
azurophil granules of neutrophils that may fullfil a quite distinct,
supportive role in neutrophil responses to tissue damage and
bacterial infections.