Neonatal Fc Receptor Binding Tolerance toward the Covalent Conjugation of Payloads to Cysteine 34 of Human Albumin Variants.

The long circulatory half-life of albumin facilitated by the interaction with the cellular recycling neonatal Fc receptor (FcRn) is utilized for drug half-life extension. FcRn engagement effects following covalent attachment of cargo to cysteine 34, however, have not been investigated. Poly(ethylene glycol) polymers were used to study the influence of cargo molecular weight on human FcRn engagement of recombinant wild type (WT) albumin and an albumin variant engineered for increased FcRn binding. Decreased affinity was observed for all conjugates; however, the engineered albumin maintained an affinity above that of unmodified wild type albumin that promotes it as an attractive drug delivery platform.

Large area protein patterning reveals nanoscale control of focal adhesion development.

Focal adhesion development in cells adherent to surface bound fibronectin presented as 200, 500, or 1000 nm diameter circular patches or as homogeneous controls is studied by fluorescence and scanning electron microscopy. Fundamental cellular processes such as adhesion, spreading, focal adhesion and stress fiber formation are shown to be dependent on the spatial distribution of ligands at this scale. Large area samples enable the study of whole cell populations and opens for new potential applications.

FAM20C-Mediated Phosphorylation of MEPE and Its Acidic Serine- and Aspartate-Rich Motif.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed in bone and teeth where it has multiple functions. The C-terminus of MEPE contains a mineral-binding, acidic serine- and aspartate-rich motif (ASARM) that is also present in other noncollagenous proteins of mineralized tissues. MEPE-derived ASARM peptides function in phosphate homeostasis and direct inhibition of bone mineralization in a phosphorylation-dependent manner. MEPE is phosphorylated by family with sequence similarity 20, member C (FAM20C), which is the main kinase phosphorylating secreted phosphoprotein. Although the functional importance of protein phosphorylation status in mineralization processes has now been well-established for secreted bone and tooth proteins (particularly for osteopontin), the phosphorylation pattern of MEPE has not been previously determined. Here we provide evidence for a very high phosphorylation level of this protein, reporting on the localization of 31 phosphoresidues in human MEPE after coexpression with FAM20C in HEK293T cells. This includes the finding that all serine residues located in the canonical target sequence of FAM20C (Ser-x-Glu) were phosphorylated, thus establishing the major target sites for this kinase. We also show that MEPE has numerous other phosphorylation sites, these not being positioned in the canonical phosphorylation sequence. Of note, and underscoring a possible important function in mineralization biology, all nine serine residues in the ASARM were phosphorylated, even though only two of these were positioned in the Ser-x-Glu sequence. The presence of many phosphorylated amino acids in MEPE, and particularly their high density in the ASARM motif, provides an important basis for the understanding of structural and functional interdependencies in mineralization and phosphate homeostasis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Osteopontin presentation affects cell adhesion-Influence of underlying surface chemistry and nanopatterning of osteopontin.

Osteopontin is a promising coating material for biomaterials, being important both in remodeling and formation of mineralized tissue and in immunological responses. We have investigated cell attachment to osteopontin adsorbed at different surface chemistries (-NH(2), -COOH, -CH(3), and bare gold) and to osteopontin presented as a nanopattern of 50 nm protein patches separated by a nonadhesive background. MDA-MB-435 cells adhere well to osteopontin presented at the hydrophilic chemistries (-NH2, -COOH, and gold) suggesting that osteopontin is presented in a functional form on these surfaces. On the amine surface, the cell attachment appears partly driven by electrostatic attraction between the positively charged substrate and the negatively charged cell membrane, whereas the spreading of the cells depends on the specific interaction with osteopontin presented at the surface. Significantly, fewer cells adhere to osteopontin presented at the methyl-terminated hydrophobic surface and the cells are less spread. On the nanopatterned osteopontin, only a very low number of cells adhered and those few attached cells showed an elongated morphology with few adhesion points to the surface. This indicates that the adhesive patches are not large enough to support stable focal contacts. The good cell attachment and spreading on the hydrophilic surfaces holds promise for osteopontin as a future coating for biomaterials.

Role of calcium phosphate nanoclusters in the control of calcification.

Calcium phosphate nanoclusters are equilibrium particles of defined chemical composition in which a core of amorphous calcium phosphate is sequestered within a shell of casein phosphopeptides. Sequence analyses and a structure prediction method were applied to secreted phosphoproteins of known importance in controlling calcification, and eight noncasein phosphoproteins were identified as containing one or more subsequences capable of forming nanoclusters. Small-angle X-ray scattering was used to confirm that a plasmin phosphopeptide of one of the identified proteins, osteopontin, formed a novel type of calcium phosphate nanocluster in which the radius of the amorphous calcium phosphate core was four times larger than is typical of casein nanoclusters. A thermodynamic treatment of nanocluster formation identified the factors of importance in determining the equilibrium size of the core, and showed how a nanocluster solution could be thermodynamically stable yet supersaturated with respect to the mineral phase of bones and teeth. It is suggested that the ability of some secreted phosphoproteins to form nanoclusters is physiologically important for the control or inhibition of calcification in soft and mineralized tissues, the extracellular matrix and a wide range of biofluids, including milk and blood.

Crystallization of calcium oxalates is controlled by molecular hydrophilicity and specific polyanion-crystal interactions.

To gain more insight into protein structure-function relationships that govern ectopic biomineralization processes in kidney stone formation, we have studied the ability of urinary proteins (Tamm-Horsfall protein, osteopontin (OPN), prothrombin fragment 1 (PTF1), bikunin, lysozyme, albumin, fetuin-A), and model compounds (a bikunin fragment, recombinant-, milk-, bone osteopontin, poly-L-aspartic acid (poly asp), poly-L-glutamic acid (poly glu)) in modulating precipitation reactions of kidney stone-related calcium oxalate mono- and dihydrates (COM, COD). Combining scanning confocal microscopy and fluorescence imaging, we determined the crystal faces of COM with which these polypeptides interact; using scanning electron microscopy, we characterized their effects on crystal habits and precipitated volumes. Our findings demonstrate that polypeptide adsorption to COM crystals is dictated first by the polypeptide's affinity for the crystal followed by its preference for a crystal face: basic and relatively hydrophobic macromolecules show no adsorption, while acidic and more hydrophilic polypeptides adsorb either nonspecifically to all faces of COM or preferentially to {100}/{121} edges and {100} faces. However, investigating calcium oxalates grown in the presence of these polypeptides showed that some acidic proteins that adsorb to crystals do not affect crystallization, even if present in excess of physiological concentrations. These proteins (albumin, bikunin, PTF1, recombinant OPN) have estimated total hydrophilicities from 200 to 850 kJ/mol and net negative charges from -9 to -35, perhaps representing a "window" in which proteins adsorb and coat urinary crystals (support of excretion) without affecting crystallization. Strongest effects on crystallization were observed for polypeptides that are either highly hydrophilic (>950 kJ/mol) and highly carboxylated (poly asp, poly glu), or else highly hydrophilic and highly phosphorylated (native OPN isoforms), suggesting that highly hydrophilic proteins strongly affect precipitation processes in the urinary tract. Therefore, the level of hydrophilicity and net charge is a critical factor in the ability of polypeptides to affect crystallization and to regulate biomineralization processes.

Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity.

Inorganic pyrophosphate (PP(i)) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P(i) in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP(i) are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP(i) on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP(i). This inhibition could be reversed by Tnap, but not if PP(i) was bound to mineral. PP(i) also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP(i) was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP(i), hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP(i) inhibits Tnap-mediated P(i) release from beta-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP(i) prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.