Axonin-1/TAG-1 mediates cell-cell adhesion by a cis-assisted trans-interaction.

The neural cell adhesion molecule axonin-1/TAG-1 mediates cell-cell interactions via homophilic and heterophilic contacts. It consists of six Ig and four fibronectin type III domains anchored to the membrane by glycosylphosphatidylinositol. The recently solved crystal structure indicates a module composed of the four N-terminal Ig domains as the contact site between trans-interacting axonin-1 molecules from apposed membranes. Here, we have tested domain-specific monoclonal antibodies for their capacity to interfere with homophilic binding in a cell aggregation assay. The results confirmed the existence of a binding region within the N-terminal Ig domains and identified a second region contributing to homophilic binding on the third and fourth fibronectin domains near the C terminus. The perturbation of each region alone resulted in a complete loss of cell aggregation, suggesting that axonin-1-mediated cell-cell contact results from a cooperative action of two homophilic binding regions. The data support that axonin-1-mediated cell-cell contact is formed by cis-assisted trans-binding. The N-terminal binding regions of axonin-1 establish a linear zipper-like string of trans-interacting axonin-1 molecules alternately provided by the two apposed membranes. The C-terminal binding regions strengthen the cell-cell contact by enhancing the expansion of the linear string into a two-dimensional array via cis-interactions. Cis-assisted trans-binding may be a basic binding mechanism common to many cell adhesion molecules.     

MDR and PRP: a comparison of methods for high-order genotype-phenotype associations

Complex diseases such as cardiovascular disease are likely due to the effects of high-order interactions among multiple genes and demographic factors. Therefore, in order to understand their underlying biological mechanisms, we need to consider simultaneously the effects of genotypes across multiple loci. Statistical methods such as multifactor dimensionality reduction (MDR), the combinatorial partitioning method (CPM), recursive partitioning (RP), and patterning and recursive partitioning (PRP) are designed to uncover complex relationships without relying on a specific model for the interaction, and are therefore well-suited to this data setting. However, the theoretical overlap among these methods and their relative merits have not been well characterized. In this paper we demonstrate mathematically that MDR is a special case of RP in which (1) patterns are used as predictors (PRP), (2) tree growth is restricted to a single split, and (3) misclassification error is used as the measure of impurity. Both approaches are applied to a case-control study assessing the effect of eleven single nucleotide polymorphisms on coronary artery calcification in people at risk for cardiovascular disease.     

Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.     

Thyroid hormone stimulates synthesis of a cardiac myosin isozyme. Comparison of the two-two-dimensional electrophoretic patterns of the cyanogen bromide peptides of cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits.

The CNBr peptides of [14C]carboxymethylated cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits have been compared using a two-dimensional electrophoretic system. The results indicated that there were extensive differences in the peptide "maps" of these heavy chains, which included differences in the distribution of radiolabeled thiol peptides. Also, the patterns of heavy chain peptides from the cardiac myosins have been compared with those produced by the heavy chain myosin isozymes from skeletal muscles. Peptide maps of heavy chains from red skeletal muscle myosin closely resembled the pattern of peptides found with cardiac myosin heavy chains from euthyroid rabbits. However, peptide maps of heavy chains from white skeletal muscle myosin were dissimilar to those of the cardiac myosin isozymes. We conclude that thyroxine administration stimulates the synthesis of a cardiac myosin isozyme with a heavy chain primary structure which is different from either of the skeletal muscle myosin isozymes.     

Measurement of waist circumference predicts coronary atherosclerosis beyond plasma adipokines

Objective:

The association of plasma adipokines beyond waist circumference (WC) with coronary artery calcification (CAC), a measure of subclinical atherosclerosis, is unknown.

Design and Methods:

Asymptomatic Caucasian individuals from two community‐based cross‐sectional studies (n = 1,285) were examined and multivariate analysis of traditional risk factors was performed, then WC and adipokines (adiponectin and leptin) were added. Incremental value of each was tested with likelihood ratio testing.

Results:

Beyond traditional risk factors, WC (Tobit regression ratio 1.69, P < 0.001) and plasma leptin (1.57, P < 0.001) but not plasma adiponectin (P = 0.75) were independently associated with CAC. In nested models, neither adiponectin (χ² = 0.76, P = 0.38) nor leptin (χ² = 1.32, P = 0.25) added value to WC beyond traditional risk factors, whereas WC added incremental value to adiponectin (χ² = 28.02, P < 0.0001) and leptin (χ² = 13.58, P = 0.0002).

Conclusion:

In the face of important biomarkers such as plasma adiponectin and leptin, WC remained a significant predictor of CAC beyond traditional risk factors underscoring the importance of WC measurement during cardiovascular risk assessment.     

The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies

The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nm range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.     

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

ApoE plays an important role in lipoprotein metabolism. This study investigated the effects of adenovirus-mediated human apoE overexpression (AdhApoE3) on sterol metabolism and in vivo reverse cholesterol transport (RCT). In wild-type mice, AdhApoE3 resulted in decreased HDL cholesterol levels and a shift toward larger HDL in plasma, whereas hepatic cholesterol content increased (P < 0.05). These effects were dependent on scavenger receptor class B type I (SR-BI) as confirmed using SR-BI-deficient mice. Kinetic studies demonstrated increased plasma HDL cholesteryl ester catabolic rates (P < 0.05) and higher hepatic selective uptake of HDL cholesteryl esters in AdhApoE3-injected wild-type mice (P < 0.01). However, biliary and fecal sterol output as well as in vivo macrophage-to-feces RCT studied with (3)H-cholesterol-loaded mouse macrophage foam cells remained unchanged upon human apoE overexpression. Similar results were obtained using hApoE3 overexpression in human CETP transgenic mice. However, blocking ABCA1-mediated cholesterol efflux from hepatocytes in AdhApoE3-injected mice using probucol increased biliary cholesterol secretion (P < 0.05), fecal neutral sterol excretion (P < 0.05), and in vivo RCT (P < 0.01), specifically within neutral sterols. These combined data demonstrate that systemic apoE overexpression increases i) SR-BI-mediated selective uptake into the liver and ii) ABCA1-mediated efflux of RCT-relevant cholesterol from hepatocytes back to the plasma compartment, thereby resulting in unchanged fecal mass sterol excretion and overall in vivo RCT.     

The effects of apolipoprotein F deficiency on high density lipoprotein cholesterol metabolism in mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20-25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/-0.9 mg/dl vs. WT: 1.2+/-0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls.