James W. Goding

Conjugation of antibodies with fluorochromes: modifications to the standard methods.

A number of modification to the standard procedures for coupling of fluorochromes to antibodies are described. The suggested procedures result in economies of time, labour and materials, and allow the reliable production of high quality conjugates. The modifications include the use of staphylococcal protein A-Sepharose for (a) a simple one-step procedure for preparation of IgG from whole serum, (b) removal of undigested IgG after pepsin treatment, and (c) concentration of dilute solutions of IgG. Many other details of coupling procedures are discussed, and modifications suggested. Optimally coupled antibodies were separated by linear salt gradient elution from DEAESephadex, and the effects of fluorescein and tetramethyl rhodamine on the antibody isoelectric point studied.

Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization

Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PPi). PPi suppresses the formation and growth of hydroxyapatite crystals. PPi is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP (Akp2) and PC-1 (Enpp1) genes. Each allele of Akp2 and Enpp1 has a measurable influence on mineralization status in vivo. Ex vivo experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PPi content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PPi concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PPi insufficiency.

Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family.

The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized. E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-beta and glucocorticoids, but the signal transduction pathways that control expression are poorly documented. NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes. In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases.

Central administration of leptin to ovariectomized ewes inhibits food intake without affecting the secretion of hormones from the pituitary gland: evidence for a dissociation of effects on appetite and neuroendocrine function.

We have studied the effect of leptin on food intake and neuroendocrine function in ovariectomized ewes. Groups (n = 5) received intracerebroventricular infusions of either vehicle or leptin (20 microg/h) for 3 days and were blood sampled over 6 h on days -1, 2, and for 3 h on day 3 relative to the onset of the infusion. The animals were then killed to measure hypothalamic neuropeptide Y expression by in situ hybridization. Plasma samples were assayed for metabolic parameters and pituitary hormones. Food intake was reduced by leptin, but did not change in controls. Leptin treatment elevated plasma lactate and nonesterified fatty acids, but did not affect glucose or insulin levels, indicating a state of negative energy balance that was met by the mobilization of body stores. Pulse analysis showed that the secretion of LH and GH was not affected by leptin treatment, nor were the mean plasma concentrations of FSH, PRL, or cortisol. Expression of messenger RNA for neuropeptide Y in the arcuate nucleus was reduced by the infusion of leptin, primarily due to reduced expression per cell rather than a reduction in the number of cells observed. Thus, the action of leptin to inhibit food intake is dissociated from neuroendocrine function. These results suggest that the metabolic effects of leptin are mediated via neuronal systems that possess leptin receptors rather than via endocrine effects.

Autosomal-Recessive Hypophosphatemic Rickets Is Associated with an Inactivation Mutation in the ENPP1 Gene

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.

Ecto‐phosphodiesterase/pyrophosphatase of lymphocytes and non‐lymphoid cells: structure and function of the PC‐1 family

Summary: Many developmentally regulated membrane proteins of lymphocytes are ecto‐enzymes, with their active sites on the external surface of the cell. These enzymes commonly have peptidase, phosphodiesterase or nucleotidase activity. Their biological roles are just beginning to be discovered. Although their expression is usually associated with particular stages of lymphoid differentiation, the same gene products are often expressed on the surface of certain non‐lymphoid cell types outside the immune system, indicating that their functions cannot be unique to lymphocytes, nor can they be ubiquitous. The plasma cell membrane protein PC‐1 (phosphodiesterase I; EC 3.1.4.1/nucleotide pyrophosphatase; EC 3.6.1.9), which was one of the first serological markers for lymphocyte subsets to be discovered, is a typical example. Within the immune system, PC‐1 is confined to plasma cells, which represent about 0.1% of lymphocytes. However, PC‐1 is also expressed on cells of the distal convoluted tubule of the kidney, chondrocytes, osteoblasts, epididymis and hepatocytes. Recent work has shown that PC‐1 is a member of a multigene family of ecto‐phosphodiesterases that currently has two other members, PD‐1α (autotaxin) and PD‐1β (B10). Within this family, the extracellular domains are highly conserved, especially around the active site. In contrast, the transmembrane and cytopiasmic domains are highly divergent. Individual members of the ecto‐phosphodiesterase family have distinct patterns of distribution in different cell types, and even within the same cell. For example, PC‐1 is present only on the basolateral surface of hepatocytes, while B10 (PD‐1β) is confined to the apical surface. Analysis of conservation and differences in the sequence of their cytoplasmic tails may illuminate intracellular tar‐getting signals. Ecto‐phosphodiesterases may play a part in diverse activities in different tissues, including recycling of nucleotides. They may also regulate the concentration of pharmacologically active extracellular compounds such as adenosine or its derivatives and ceil motility. Some members may modulate local concentrations of pyrophosphate, and hence influence calcification in bone and cartilage.

Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification

OBJECTIVE Elevated cartilage inorganic pyrophosphate (PPi) production and PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity are strongly linked with aging-related cartilage calcification in meniscal and articular cartilages. We hypothesized that there were divergent relationships of 3 NTPPPH isozymes with cartilage matrix calcification and sought to identify them. METHODS We studied knee medial meniscal expression in situ of 3 NTPPPH isozymes of the phosphodiesterase nucleotide pyrophosphatase (PDNP) family: plasma cell membrane glycoprotein 1 (PC-1, or PDNP1), autotaxin (ATX, or PDNP2), and B10/PDNP3. We also used complementary DNA transfection to assess differential functions in matrix calcification of each NTPPPH isozyme in vitro in meniscal cells. RESULTS We observed diffuse cell-associated ATX and B10/PDNP3 expression in central (chondrocytic) and, to a lesser degree, peripheral (fibroblastic) regions of normal, degenerative uncalcified, and degenerative calcified menisci. In contrast, PC-1 expression was only robust at sites of apoptotic cells and calcification in central regions of degenerative menisci. Only PC-1 was abundant at the perimeter of meniscal cells and in association with meniscal cell-derived matrix vesicles (MVs). Because each PDNP-family isozyme was expressed by cells near calcifications, we transfected the isozymes in nonadherent knee meniscal cells cultured with ascorbic acid, beta-glycerophosphate, and dexamethasone supplementation to stimulate them to calcify the matrix. PC-1, but not ATX or B10/PDNP3, consistently promoted increased MV NTPPPH, MV-associated PPi, and extracellular PPi. PC-1 also increased matrix calcification (with hydroxyapatite crystals) by meniscal cells. ATX uniquely induced alkaline phosphatase activity, but promoted only moderately increased matrix calcification. CONCLUSION We identified divergent effects of 3 PDNP-family NTPPPH isozymes on meniscal cell matrix calcification. Increased expression of PC-1 is both a marker and a potential pathogenic factor for knee meniscal cartilage matrix calcification.

Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.

OBJECTIVE Increased nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity in chondrocytes is associated with cartilage matrix inorganic pyrophosphate (PPi) supersaturation in chondrocalcinosis. This study compared the roles of the transforming growth factor beta (TGFbeta)-inducible plasma cell membrane glycoprotein-1 (PC-1) and the closely related B10 NTPPPH activities in chondrocyte PPi metabolism. METHODS NTPPPH expression was studied using reverse transcriptase-polymerase chain reaction and Western blotting. Transmembrane PC-1 (tmPC-1), water-soluble secretory PC-1 (secPC-1), and transmembrane B10 were expressed by adenoviral gene transfer or plasmid transfection, and expression of PPi was assessed in cultured articular chondrocytes and immortalized NTPPPH-deficient costal chondrocytes (TC28 cells). RESULTS PC-1 and B10 messenger RNA were demonstrated in articular cartilages in situ, in untreated cultured normal articular chondrocytes, and in TC28 cells. Expression of tmPC-1 and secPC-1, but not B10, rendered the NTPPPH-deficient TC28 cells able to increase expression of extracellular PPi, with or without addition of TGFbeta (10 ng/ml) to the media. More plasma membrane NTPPPH activity was detected in cells transfected with tmPC-1 than in cells transfected with B10. Furthermore, confocal microscopy with immunofluorescent staining of articular chondrocytes confirmed preferential plasma membrane localization of PC-1, relative to B10. Finally, both PC-1 and B10 increased the levels of intracellular PPi, but PC-1 and B10 appeared to act principally in different intracellular compartments (Golgi and post-Golgi versus pre-Golgi, respectively). CONCLUSION PC-1 and B10 NTPPPH activities were not redundant in chondrocytes. Although increased PC-1 and B10 expression caused elevations in intracellular PPi, the major effects of PC-1 and B10 were exerted in distinct subcellular compartments. Moreover, PC-1 (transmembrane and secreted), but not B10, increased the levels of extracellular PPi. Differential expression of PC-1 and B10 could modulate cartilage mineralization in degenerative joint diseases.

Matrix Vesicle Plasma Cell Membrane Glycoprotein-1 Regulates Mineralization by Murine Osteoblastic MC3T3 Cells†

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)‐generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC‐1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in “tiptoe walking” (ttw) mice. Thus, we tested the hypothesis that PC‐1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC‐1 mRNA and produced hydroxyapatite deposits at 12–14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor‐β and 1,25‐dihydroxyvitamin D3 increased PC‐1 and NTPPPH in matrix vesicles (MVs). Because PC‐1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2‐ to 6‐fold more PC‐1. PC‐1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC‐1. Furthermore, MC3T3 cells transfected with PC‐1 deposited ∼80–90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC‐1. ATP‐dependent45Ca precipitation by MVs from cells overexpressing active PC‐1 was comparably diminished. Thus, regulation of PC‐1 controls the PPi content and function of osteoblast‐derived MVs and matrix hydroxyapatite deposition. PC‐1 may provide a novel therapeutic target in certain disorders of bone mineralization.

Ecto-enzymes: physiology meets pathology.

Ecto‐enzymes are catalytic membrane proteins with their active sites outside the cell. They include cholinesterase, which inactivates acetylcholine, and angiotensin‐converting enzyme, which converts angiotensin I to biologically active angiotensin II, and numerous other peptidases, transpeptidases, nucleotidases, phosphodiesterases, and phosphatases. Many CD antigens of leukocytes are ecto‐enzymes; some CD antigens for which no function is currently known are probably ectoenzymes. Expression is highly regulated and correlated with differentiation and activation. Some are highly restricted in distribution; others are ubiquitous. Many are shared between leukocytes and non‐hematogenous cells. Biological functions appear to depend on the type and location of the cell in which expression occurs, and include recycling of nutrients, local control of response to cytokines and hormones, bone formation, cell mobility, invasion, and metastasis. Many novel regulatory functions of ecto‐enzymes remain to be discovered, and may reveal new mechanisms of local extracellular control of cellular function. J. Leukoc. Biol. 67: 285–311; 2000.