The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α-or β-mannosyl-terminating asparagine (N )-linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue-and subcellular-specific expression of PMPs within invertebrates and plants, often comprising multi-α1,3/6-fucosylation and β1,2-xylosylation amongst other glycan modifications and non-glycan substitutions e.g. O-methylation. Vertebrates and protists express less-heterogeneous PMPs typically only comprising variable core fucosylation of bi-and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less-truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue-wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell-cell communication and host-pathogen/symbiont interactions. In most PMP-producing organisms, including humans, the N -acetyl-β-hexosaminidase isoenzymes and linkage-specific α-mannosidases are glycoside hydrolases critical for generating PMPs via N -acetylglucosaminyltransferase I (GnT-I)-dependent and GnT-I-independent truncation pathways. However, the identity and structure of many species-specific PMPs in eukaryotes, their biosynthetic routes, strong tissue-and development-specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP-recognising lectins across a variety of healthy and N -acetyl-β-hexosaminidase-deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongs...

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called ‘targeting-by-timing’. Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation and N- and O-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules (AG), whereas the later-formed specific and gelatinase granules (SG and GG) and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immuno-blotting and –histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils and their complexity increased progressively from AG to SG and then to GG, suggesting temporal changes in the glycosylation machinery indicative of ‘glycosylation-by-timing’ during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step towards a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

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