Lysyl hydroxylases catalyze hydroxylation of collagen lysines, and sustain essential roles in extracellular matrix (ECM) maturation and remodeling. Malfunctions in these enzymes cause severe connective tissue disorders. Human lysyl hydroxylase 3 (LH3/PLOD3) bears multiple enzymatic activities, as it catalyzes collagen lysine hydroxylation and also their subsequent glycosylation. Our understanding of LH3 functions is currently hampered by lack of molecular structure information. Here, we present high resolution crystal structures of full-length human LH3 in complex with cofactors and donor substrates. The elongated homodimeric LH3 architecture shows two distinct catalytic sites at the N- and C-terminal boundaries of each monomer, separated by an accessory domain. The glycosyltransferase domain displays distinguishing features compared to other known glycosyltransferases. Known disease-related mutations map in close proximity to the catalytic sites. Collectively, our results provide a structural framework characterizing the multiple functions of LH3, and the molecular mechanisms of collagen-related diseases involving human lysyl hydroxylases.

BackgroundPathogenic PLOD3 variants cause a connective tissue disorder (CTD) that has been described rarely. We further characterise this CTD and propose a clinical diagnostic label to improve recognition and diagnosis of PLOD3-related disease.MethodsReported PLOD3 phenotypes were compared with known CTDs utilising data from three further individuals from a consanguineous family with a homozygous PLOD3 c.809C>T; p.(Pro270Leu) variant. PLOD3 mRNA expression in the developing embryo was analysed for tissue-specific localisation. Mouse microarray expression data were assessed for phylogenetic gene expression similarities across CTDs with overlapping clinical features.ResultsKey clinical features included ocular abnormalities with risk for retinal detachment, sensorineural hearing loss, reduced palmar creases, finger contractures, prominent knees, scoliosis, low bone mineral density, recognisable craniofacial dysmorphisms, developmental delay and risk for vascular dissection. Collated clinical features showed most overlap with Stickler syndrome with variable features of Ehlers-Danlos syndrome (EDS) and epidermolysis bullosa (EB). Human lysyl hydroxylase 3/PLOD3 expression was localised to the developing cochlea, eyes, skin, forelimbs, heart and cartilage, mirroring the clinical phenotype of this disorder.ConclusionThese data are consistent with pathogenic variants in PLOD3 resulting in a clinically distinct Stickler-like syndrome with vascular complications and variable features of EDS and EB. Early identification of PLOD3 variants would improve monitoring for comorbidities and may avoid serious adverse ocular and vascular outcomes.

During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.

PLOD genes encode for procollagen lysyl hydroxylase enzymes (LH/PLOD), a family of proteins essential for collagen biosynthesis. Several mutations affect these genes, causing severe disorders, such as Ehlers‐Danlos and Bruck syndrome, as well a connective tissue disease with phenotype resembling osteogenesis imperfecta caused by lack of LH3 functions. The recently determined three‐dimensional (3D) structures of the full‐length human LH3/PLOD3 isoform, together with the structure of a fragment of a viral LH/PLOD homolog, are now allowing molecular mapping of the numerous disease‐causing mutations, providing insights often suitable for the interpretation of the resulting disease phenotypes. However, the added value of molecular structure interpretation is affected by the limited accessibility of complex molecular data to scientific communities lacking direct expertise in structural biology. In this work, we present a Structurally‐integrated database for Mutations of PLOD genes (SiMPLOD), a publicly‐available manually‐curated online database with an embedded molecular viewer interface for the visualization and interpretation of LH/PLOD mutations on available molecular models. Each SiMPLOD entry is accompanied by manual annotations extrapolated from literature references and comments about the localization of the amino acid variants on the molecular structure. Additional links to the appropriate online resources for clinically‐relevant as well as biochemical data are also provided in a standardized format. The web application is available at http://fornerislab.unipv.it/SiMPLOD. © 2019 American Society for Bone and Mineral Research.

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