Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions*.

Abstract

Human neutrophil elastase (HNE) is an important -glycosylated serine protease in the innate immune system, but the structure and immune-modulating functions of HNE-glycosylation remain undescribed. Herein, LC-MS/MS-based glycan, glycopeptide and glycoprotein profiling were utilized to first determine the heterogeneous -glycosylation of HNE purified from neutrophil lysates and then from isolated neutrophil granules of healthy individuals. The spatiotemporal expression of HNE during neutrophil activation and the biological importance of its-glycosylation were also investigated using immunoblotting, cell surface capture, native MS, receptor interaction, protease inhibition, and bacteria growth assays. Site-specific HNE glycoprofiling demonstrated that unusual paucimannosidic -glycans, particularly Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ, predominantly occupied Asn124 and Asn173. The equally unusual core fucosylated monoantenna complex-type-sialoglycans also decorated these two fully occupied sites. In contrast, the mostly unoccupied Asn88 carried nonfucosylated paucimannosidic -glycans probably resulting from low glycosylation site solvent accessibility. Asn185 was not glycosylated. Subcellular- and site-specific glycoprofiling showed highly uniform-glycosylation of HNE residing in distinct neutrophil compartments. Stimulation-induced cell surface mobilization demonstrated a spatiotemporal regulation, but not cell surface-specific glycosylation signatures, of HNE in activated human neutrophils. The three glycosylation sites of HNE were located distal to the active site indicating glycan functions other than interference with HNE enzyme activity. Functionally, the paucimannosidic HNE glycoforms displayed preferential binding to human mannose binding lectin compared with the HNE sialoglycoforms, suggesting a glycoform-dependent involvement of HNE in complement activation. The heavily -glycosylated HNE protease inhibitor, α1-antitrypsin, displayed concentration-dependent complex formation and preferred glycoform-glycoform interactions with HNE. Finally, both enzymatically active HNE and isolated HNE-glycans demonstrated low micromolar concentration-dependent growth inhibition of clinically-relevant , suggesting some bacteriostatic activity is conferred by the HNE-glycans. Taken together, these observations support that the unusual HNE -glycosylation, here reported for the first time, is involved in modulating multiple immune functions central to inflammation and infection.

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

“…Paucimannosylation is a common modification of proteins expressed by lower organisms, but our recent reports indicate that higher organisms including humans also express paucimannosidic proteins albeit in a more tissue‐restricted and physiology‐dependent manner . Paucimannosylation appears particularly central to the human innate immune system as supported by our previously published data documenting that PMGs are carried by biologically important proteins still displaying bioactivity and structural integrity expressed by resting and activated human neutrophils and macrophages . The findings presented herein documenting that PMGs also serve as signatures of human cancers add to our knowledge of this understudied N ‐glycan type, but at the same time prompts several questions, some of which are discussed below to encourage and stimulate further activity in this research field.…”

“…However, it is likely that the PMGs identified from proteins extracted from the tumor tissues originate from multiple cell types including directly from the cancer cells as well as from the cancer‐associated stroma, adipocytes, immune cells, and necrotic cells forming the heterogeneous tumor micro‐environment . In support, M2Fa and to a lesser extent M3Fa, which in this study were found to be key PMGs of the tumor tissues, have repeatedly been reported to be abundant glycans carried by proteins expressed by neutrophils and macrophages . Neutrophils and macrophages are important innate immune cells that are known to reside in and modulate the tumor micro‐environment as tumor‐associated neutrophils (TANs) and macrophages (TAMs) .…”

“…Our recent summary of the literature supports that humans, similar to most other eukaryotic species, produce proteins modified by paucimannosidic glycans (PMGs) comprising simple monosaccharide compositions, Man(M) 1‐3 GlcNAc 2 with a variable presence of core fucosylation, Fuc(F) 0‐1 albeit in a more tissue‐specific and physiology‐dependent manner than the lower organisms, for example, invertebrates and plants that constitutively express paucimannosidic proteins . Our detailed literature survey also supports that protein paucimannosylation plays important functions in the human innate immune system, in infectious processes, and cellular development, accurately mirroring the biological environments where most human PMG‐modified proteins to date have been observed …”

“…A less studied type of human N ‐glycosylation, known as protein paucimannosylation, is receiving growing attention in human glycobiology . Our recent summary of the literature supports that humans, similar to most other eukaryotic species, produce proteins modified by paucimannosidic glycans (PMGs) comprising simple monosaccharide compositions, Man(M) 1‐3 GlcNAc 2 with a variable presence of core fucosylation, Fuc(F) 0‐1 albeit in a more tissue‐specific and physiology‐dependent manner than the lower organisms, for example, invertebrates and plants that constitutively express paucimannosidic proteins .…”

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function

“…Differences in primary structure do not account for the observed differences in N-glycan processing: the CD16a polypeptide sequence and presence of the coexpressed Fc e receptor g-chain were the same in both NK cells and that used in HEK293F cells to express frCD16a. Cell-type specific differences in the glycosylation of a specific protein are known (40)(41)(42)(43)(44) and are justifiable given the complexity of gene expression and activity for glycan-processing enzymes in different tissues (45)(46)(47)(48)(49)(50).…”