The matrix metalloproteinases (MMPs) form an enzyme family of which gelatinase B (MMP-9) represents the largest and most complex member. We focus here on the biochemical properties, regulation, and functions of gelatinase B. The tight regulation of gelatinase B activity is highly complex and is established at five different levels. The transcription of the gelatinase B-gene depends on various cis-elements in its gene promotor and is induced or repressed by a large variety of soluble factors, including cytokines, growth factors, and hormones and by cellular contacts acting through specific signaling pathways. The specific regulation of its secretion occurs in cells storing gelatinase B in granules. After secretion, progelatinase B must be activated through an activation network. The enzyme activity is further regulated by inhibition and by other mechanisms, such as fine-tuning and stabilization by glycosylation. The ability of gelatinase B to degrade components of the extracellular matrix and to regulate the activity of a number of soluble proteins confers an important role in various physiological and pathological processes. These include reproduction, growth, development, inflammation, and vascular and proliferative diseases.

Research on matrix metalloproteinases (MMPs) and in particular on gelatinase B, alias MMP-9, has grown exponentially in the decade 2003–2012. Structural details about flexibility of MMP-9 monomers, together with glycosylation, oligomerization, heterogeneity and instability of the wildtype enzyme explain why crystallography experiments have not yet been successful for the intact enzyme. MMP-9 may be viewed as a multidomain enzyme in which the hemopexin, the O-glycosylated and the catalytic domains yield support for attachment, articulation and catalysis, respectively. The stepwise proteolytic activation of the inactive zymogen into a catalytically active form becomes gradually better understood. Priming of activation by MMP-3 may be executed by meprins that destabilize the interaction of the aminoterminus with the third fibronectin repeat. Alternatively, autocatalytic activation may occur in the presence of molecules that tightly bind to the catalytic site and that push the cystein residue in the prodomain away from the catalytic zinc ion. Thanks to the development of degradomics technologies, substrate repertoires of MMP-9 have been defined, but it remains a challenge to determine and prove which substrates are biologically relevant. The substrate repertoire has been enlarged from extracellular to membrane-bound and efficient intracellular substrates, such as crystallins, tubulins and actins. Biological studies of MMP-9 have tuned the field from being primarily cancer-oriented towards vascular and inflammatory research. In tumor biology, it has been increasingly appreciated that MMP-9 from inflammatory cells, particularly neutrophils, co-determines prognosis and outcome. Aside from the catalytic functions executed by aminoterminal domains of MMP-9, the carboxyterminal hemopexin (PEX) domain of gelatinase B exerts non-catalytic anti-apoptotic signaling effects. The recognition that gelatinase B is induced by many pro-inflammatory cytokines, whereas its inhibitors are increased by anti-inflammatory cytokines, has generated interest to target MMP-9 in acute lethal conditions, such as bacterial meningitis, sepsis and endotoxin shock, and in acute exacerbations of chronic diseases. Previously described transcriptional regulation of MMP-9 is complemented by epigenetic checkpoints, including histone modifications and microRNAs. Because activation of proMMP-9 may be executed by other MMPs, the therapeutic dogma that MMP inhibitors need to be highly selective may be keyed down for the treatment of life-threatening conditions. When inflammation and MMP-9 fulfill beneficial functions to clear damaging protein complexes, such as in systemic autoimmune diseases, therapeutic MMP inhibition has to be avoided. In Mmp9 gene knockout mice, specific spontaneous phenotypes emerged with effects on the skeletal, reproductive and nervous systems. These findings not only have clinical correlates in bone growth and fertility, but also stimulate research on the roles of MMPs and MMP-9 in endocrinology, immunology and the neurosciences. Mmp9-deficient mice are valuable tools to define MMP-9 substrates in vivo and to study the role of this enzyme in animal models of inflammatory, vascular, neoplastic and degenerative diseases. Future challenges include solving the crystal structure, definition of the functions of covalent oligomers and heteromers in biology and pathology, life-imaging of MMP-9 activity, substrate determination in situ and the study of inhibitor effects on fertility, cancer and inflammation and in neurobiology and regenerative medicine. Such studies will better define conditions in which inhibition of MMP-9 is beneficial or has to be avoided.

The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucin-type O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosac-charides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE^−/− mice prevent collateral host tissue damage.