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Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC–MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics.

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6–12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy., Graphical abstract, Human-mouse cross-reactive monoclonal antibodies (mAbs) inhibiting the vascular endothelial growth factor (VEGF) receptor (Flt-1) with high affinity and specificity were developed. In the mdx mouse model of Duchenne muscular dystrophy (DMD), the mAbs increased VEGF levels, vascularization, and muscle function and decreased fibrosis, suggesting potential therapeutic benefit for DMD.

For three decades, the lipoprotein lipase (LPL)-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and ultraviolet resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short alpha-helix, with two tryptophans projecting into the antigen recognition site. A proline substitution in the alpha-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

Alpha galactosidase (a-Gal) is an acidic hydrolase that plays a critical role in hydrolyzing the terminal alpha-galactoyl moiety from glycolipids and glycoproteins. There are over a hundred mutations reported for the GLA gene that encodes a-Gal that result in reduced protein synthesis, protein instability, and reduction in function. The deficiencies of a-Gal can cause Fabry disease, a rare lysosomal storage disorder (LSD) caused by the failure to catabolize alpha-d-galactoyl glycolipid moieties. The current standard of care for Fabry disease is enzyme replacement therapy (ERT) where the purified recombinant form of human a-Gal is given to patients. The manufacture of a-Gal is currently performed utilizing traditional large-scale chromatography processes. Developing an affinity resin for the purification of a-Gal would reduce the complexity of the manufacturing process, reduce costs, and potentially produce a higher quality a-Gal. After the evaluation of many small molecules, a commercially available small molecule biomimetic, N-5-Carboxypentyl-1-deoxygalactonojirimycin (N5C-DGJ), was utilized for the development of a novel small molecule biomimetic affinity resin for a-Gal. Affinity purified a-Gal demonstrated a purity greater than 90%, exhibited expected thermal stability and specific activity. Complementing this affinity purification is the development of an elution buffer system that confers an increased thermal stability to a-Gal. The N5C-DGJ affinity resin tolerated sodium hydroxide sanitization with no loss of binding capacity, making it amenable to large scale purification processes and potential use in manufacturing. This novel method for purifying the challenging a-Gal enzyme can be extended to other enzyme replacement therapies.

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

Follistatin (FS) is an important regulatory protein, a natural antagonist for transforming growth factor-β family members activin and myostatin. The diverse biologic roles of the activin and myostatin signaling pathways make FS a promising therapeutic target for treating human diseases exhibiting inflammation, fibrosis, and muscle disorders, such as Duchenne muscular dystrophy. However, rapid heparin-mediated hepatic clearance of FS limits its therapeutic potential. We targeted the heparin-binding loop of FS for site-directed mutagenesis to improve clearance parameters. By generating a series of FS variants with one, two, or three negative amino acid substitutions, we demonstrated a direct and proportional relationship between the degree of heparin-binding affinity in vitro and the exposure in vivo. The triple mutation K(76,81,82)E abolished heparin-binding affinity, resulting in ∼20-fold improved in vivo exposure. This triple mutant retains full functional activity and an antibody-like pharmacokinetic profile, and shows a superior developability profile in physical stability and cell productivity compared with FS variants, which substitute the entire heparin-binding loop with alternative sequences. Our surgical approach to mutagenesis should also reduce the immunogenicity risk. To further lower this risk, we introduced a novel glycosylation site into the heparin-binding loop. This hyperglycosylated variant showed a 10-fold improved exposure and decreased clearance in mice compared with an IgG1 Fc fusion protein containing the native FS sequence. Collectively, our data highlight the importance of improving pharmacokinetic properties by manipulating heparin-binding affinity and glycosylation content and provide a valuable guideline to design desirable therapeutic FS molecules.

Lipoprotein lipase (LPL), identified in the 1950s, has been studied intensively by biochemists, physiologists, and clinical investigators. These efforts uncovered a central role for LPL in plasma triglyceride metabolism and identified LPL mutations as a cause of hypertriglyceridemia. By the 1990s, with an outline for plasma triglyceride metabolism established, interest in triglyceride metabolism waned. In recent years, however, interest in plasma triglyceride metabolism has awakened, in part because of the discovery of new molecules governing triglyceride metabolism. One such protein—and the focus of this review—is GP IHBP1, a protein of capillary endothelial cells. GPIHBP1 is LPL’s essential partner: it binds LPL and transports it to the capillary lumen; it is essential for lipoprotein margination along capillaries, allowing lipolysis to proceed; and it preserves LPL’s structure and activity. Recently, GPIHBP1 was the key to solving the structure of LPL. These developments have transformed the models for intravascular triglyceride metabolism.

Significance Lipoprotein lipase (LPL) plays a central role in plasma lipid metabolism, hydrolyzing the triglycerides in lipoproteins and releasing fatty acid nutrients for vital tissues. LPL is synthesized by adipocytes and myocytes and secreted into the interstitial spaces, whereupon it is captured by an endothelial cell protein, GPIHBP1, and transported to its site of action within the capillary lumen. For decades, LPL has been assumed to be a homodimer, with dimerization thought to be required for both secretion and catalytic activity. In the present study, we revisited the notion that LPL is a homodimer. Our findings indicate that LPL and GPIHBP1-bound LPL are active in a monomeric state., Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

Mucopolysaccharidosis III B (MPS III-B) is a rare lysosomal storage disorder caused by deficiencies in Alpha-N-acetylglucosaminidase (NAGLU) for which there is currently no cure, and present treatment is largely supportive. Understanding the structure of NAGLU may allow for identification of novel therapeutic targets for MPS III-B. Here we describe the first crystal structure of human NAGLU, determined to a resolution of 2.3 A. The crystal structure reveals a novel homotrimeric configuration, maintained primarily by hydrophobic and electrostatic interactions via domain II of three contiguous domains from the N- to C-terminus. The active site cleft is located between domains II and III. Catalytic glutamate residues, E316 and E446, are located at the top of the (α/β)8 barrel structure in domain II. We utilized the three-dimensional structure of NAGLU to map several MPS III-B mutations, and hypothesize their functional consequences. Revealing atomic level structural information about this critical lysosomal enzyme paves the way for the design of novel therapeutics to target the underlying causes of MPS III-B.

Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1’s acidic domain preserves LPL structure and activity, we crystallized an LPL–GPIHBP1 complex and solved its structure. GPIHBP1’s LU domain binds to LPL’s C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1’s acidic domain was not defined in the electron density map but was positioned to interact with LPL’s large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL–GPIHBP1 structure provides insights into mutations causing chylomicronemia.

The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1-14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization.

Fabry disease is a lysosomal storage disorder caused by mutations in the GLA gene that encodes for the lysosomal enzyme α-galactosidase A (α-Gal A). Reduced or absent α-Gal A activity leads to substrate accumulation and deleterious effects in multiple organs. Migalastat is a pharmacological chaperone that may stabilize the enzyme in specific GLA variants, considered amenable, assisting enzyme trafficking to lysosomes and thus increasing enzyme activity. Using a good laboratory practice (GLP)-validated human embryonic kidney cell (HEK)-based (GLP-HEK) amenability assay established during the clinical development of migalastat, approximately one-third of GLA variants are reported to be amenable to migalastat. On the basis of this biochemical amenability, migalastat is approved for use in patients with specific GLA variants. In this study, the reproducibility of the amenability assay was assessed by evaluation of 59 GLA variants for α-Gal A activity in the presence and absence of migalastat. As for the GLP-HEK assay, variants were considered amenable when there was both an absolute increase in enzyme activity of ≥3% wild-type and a relative increase in enzyme activity ≥1.2 fold over baseline following incubation with migalastat. Six of the 59 variants tested here did not match the classification of amenability reported using the GLP-HEK assay. Linear regression and Bland-Altman analyses, comparing data from all variants with and without migalastat, provided additional evidence for a lack of assay reproducibility. Data from the GLP-HEK assay (and the resulting classification of amenability) can determine treatment strategy and, ultimately, patient outcomes, so discrepancies between amenability assay data could be a cause for concern for physicians managing patients with Fabry disease.

The neuronal protein FE65 functions in brain development and Amyloid Precursor Protein (APP) signaling through its interaction with the mammalian enabled (Mena) protein and APP, respectively. The recognition of short polyproline sequences in Mena by the FE65 WW domain plays a central role in axon guidance and neuronal positioning in the developing brain. We have determined the crystal structures of the human FE65 WW domain (residues 253–289) in the apo form and bound to the peptides PPPPPPLPP and PPPPPPPPPL, which correspond to human Mena residues 313–321 and 347–356, respectively. The FE65 WW domain contains two parallel ligand-binding grooves, XP (formed by residues Y269 and W280) and XP2 (formed by Y269 and W271). Both Mena peptides adopt a polyproline helical II conformation and bind to the WW domain in a forward (N–C) orientation through selection of the PPPPP motif by the XP and XP2 grooves. This mode of ligand recognition is strikingly similar to polyproline interaction with SH3 domains. Importantly, comparison of the FE65 WW structures in the apo and liganded forms shows that the XP2 groove is formed by an induced-fit mechanism that involves movements of the W271 and Y269 side chains upon ligand binding. These structures elucidate the molecular determinants underlying polyproline ligand selection by the FE65 WW domain and provide a framework for the design of small molecules that would interfere with FE65 WW–ligand interaction and modulate neuronal development and APP signaling.

Transglutaminases (TGases) are Ca2+-dependent enzymes capable of catalysing transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGases have been described in mammals, and two of them (types 2 and 3) are regulated by GTP/ATP. TGase2 hydrolyses GTP and is therefore a bifunctional enzyme. In the present study, we report that TGase5 is also regulated by nucleotides. We have identified the putative TGase5 GTP-binding pocket by comparative amino acid sequence alignment and homology-derived three-dimensional modelling. GTP and ATP inhibit TGase5 cross-linking activity in vitro, and Ca2+ is capable of completely reversing this inhibition. In addition, TGase5 mRNA is not restricted to epidermal tissue, but is also present in different adult and foetal tissues, suggesting a role for TGase5 outside the epidermis. These results reveal the reciprocal actions of Ca2+ and nucleotides with respect to TGase5 activity. Taken together, these results indicate that TGases are a complex family of enzymes regulated by calcium, with at least three of them, namely TGase2, TGase3 and TGase5, also being regulated by ATP and GTP.

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.