The Propeptide Binding Site of the Bovine γ-Glutamyl Carboxylase

gamma-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the gamma-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the gamma-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the gamma-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine gamma-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum

A Conserved Region of Human Vitamin K-dependent Carboxylase between Residues 393 and 404 Is Important for Its Interaction with the Glutamate Substrate

Certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their gamma-glutamyl carboxylase causing impaired glutamate binding. The sequence surrounding Leu394 is similar in all known carboxylases, suggesting that the region is functionally important. To test this hypothesis we made the following mutant enzymes: W390A, Y395A, S398A, W399A, and H404A. We purified the enzymes and corrected the activity measurements for active enzyme concentration. Carboxylases W390A, S398A, and H404A had activities similar to that of wild type; however, Y395A and W399A had lower activities than did wild type. In the following descriptions we include our previously reported results for L394R. Kinetic studies with the substrate FLEEL, revealed Km values of 0.5 (wild type), 6.5 (L394R), 15 (Y395A), and 24 (W399A) mm. The kcat values relative to wild type were 51% (L394R), 1% (Y395A), and 2% (W399A). The kcat/Km values were 24-fold (L394R) and >2000-fold lower for Y395A and W399A than for wild-type carboxylase. Inhibition of FLEEL carboxylation by the competitive inhibitor, Boc-mEEV, gave Ki values of 0.013 (wild type), 1.4 (L394R), 2.1 (Y395A), and >5 (W399A) mm. The Y395A propeptide affinity was similar to that of wild type, but those of L394R and W399A were 16-22-fold less than that of wild type. Results of kinetic studies with a propeptide-containing substrate were consistent with results of propeptide binding and FLEEL kinetics. Although propeptide and vitamin K binding in some mutants were affected, our data provide compelling evidence that glutamate recognition is the primary function of the conserved region around Leu394

Maternal Humoral Immune Correlates of Peripartum Transmission of Clade C HIV-1 in the Setting of Peripartum Antiretrovirals

ABSTRACT Despite the widespread use of antiretrovirals (ARV), more than 150,000 pediatric HIV-1 infections continue to occur annually. Supplemental strategies are necessary to eliminate pediatric HIV infections. We previously reported that maternal HIV envelope-specific anti-V3 IgG and CD4 binding site-directed antibodies, as well as tier 1 virus neutralization, predicted a reduced risk of mother-to-child transmission (MTCT) of HIV-1 in the pre-ARV era U.S.-based Women and Infants Transmission Study (WITS) cohort. As the majority of ongoing pediatric HIV infections occur in sub-Saharan Africa, we sought to determine if the same maternal humoral immune correlates predicted MTCT in a subset of the Malawian Breastfeeding, Antiretrovirals, and Nutrition (BAN) cohort of HIV-infected mothers ( n = 88, with 45 transmitting and 43 nontransmitting). Women and infants received ARV at delivery; thus, the majority of MTCT was in utero (91%). In a multivariable logistic regression model, neither maternal anti-V3 IgG nor clade C tier 1 virus neutralization was associated with MTCT. Unexpectedly, maternal CD4 binding-site antibodies and anti-variable loop 1 and 2 (V1V2) IgG were associated with increased MTCT, independent of maternal viral load. Neither infant envelope (Env)-specific IgG levels nor maternal IgG transplacental transfer efficiency was associated with transmission. Distinct humoral immune correlates of MTCT in the BAN and WITS cohorts could be due to differences between transmission modes, virus clades, or maternal antiretroviral use. The association between specific maternal antibody responses and in utero transmission, which is distinct from potentially protective maternal antibodies in the WITS cohort, underlines the importance of investigating additional cohorts with well-defined transmission modes to understand the role of antibodies during HIV-1 MTCT

Effect of Vitamin K-dependent Protein Precursor Propeptide, Vitamin K Hydroquinone, and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO3, on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation

Expression and Characterization of the Naturally Occurring Mutation L394R in Human γ-Glutamyl Carboxylase

Patients with mutation L394R in gamma-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide

Polytopic membrane protein folding at L17 in the ribosome tunnel initiates cyclical changes at the translocon

Interaction between L17 in the ribosome tunnel and folded nascent chain transmembrane segments during multi-spanning membrane protein synthesis triggers structural rearrangements in the ribosome that cause switching between cytosolic and ER lumenal targeting of the growing polypeptide

Measurement of true ileal phosphorus digestibility in feed ingredients for poultry : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Poultry Science at Institute of Veterinary, Animal and Biomedical Sciences (IVABS), Massey University, Palmerston North, New Zealand

Global interest in improving the utilisation of phosphorus (P) by poultry has recently increased due to concerns over environmental pollution through excess P excretion, depletion of non-renewable inorganic phosphate deposits, and increasing price of inorganic phosphate supplements. Use of a sound criterion, preferably based on P digestibility, to assess P availability is needed to enable greater efficiency of utilisation of dietary P. No established methodology is currently available to measure the true digestible P contents in common feed ingredients for poultry. The first experiment of this thesis (Chapter 3) investigated the effects of dietary calcium (Ca) concentrations (6, 9 and 12 g/kg) on the digestibility of P, Ca, nitrogen, fat and starch in different intestinal segments and on the apparent metabolisable energy of diets in young broiler chickens. The results showed that the digestion of P and Ca was completed by upper ileum and jejunum, respectively. The site of digestion of P and nitrogen was found to shift depending on the dietary Ca concentrations. The digestibility coefficients of P in low, normal and high Ca diets at the lower ileum were determined to be 0.417, 0.379 and 0.325, respectively. The overall data showed that increasing dietary Ca concentrations negatively influenced the digestion of P, nitrogen and fat, but had no effect on those of Ca, starch and apparent metabolisable energy. The second experiment (Chapter 4) was conducted to determine endogenous losses of P and Ca in broiler chickens. The data showed that the ileal endogenous P losses in birds differed depending on the methodology employed. The ileal endogenous flow of P in birds fed P-free, gelatin-based and casein-based diets were 25, 104 and 438 mg/kg dry matter intake (DMI), respectively. Ileal endogenous flow of Ca in birds fed casein-based diet was estimated to be 321 mg/kg DMI. The next three experiments (Chapters 5, 6 and 7) investigated the potential usefulness of regression method to evaluate true ileal P digestibility of seven feed ingredients. True ileal P digestibility coefficients of maize, canola meal, wheat, sorghum, soybean meal and maize-distiller‟s dried grains with solubles for broiler chickens were determined to be 0.676, 0.469, 0.464, 0.331, 0.798 and 0.727, respectively. For plant-based ingredients, the determined true digestible P values were consistently higher than corresponding non-phytate P values (Maize, 1.72 vs. 0.75; canola meal, 4.55 vs. 2.82; wheat, 1.49 vs. 1.11; sorghum, 0.78 vs. 0.55; soybean meal, 5.16 vs. 2.15; maize-distiller‟s dried grains with solubles, 5.94 vs. 4.36 g/kg, as fed ii basis, respectively). Phytate P in maize (54.25%), soybean meal (69.7%) and maize- distiller‟s dried grains with solubles (41.5%) were well digested by broilers compared to canola meal (25.2%), wheat (18.1%) and sorghum (13.0%). True ileal P digestibility coefficients of three meat and bone meal (MBM) samples ranged from 0.420 to 0.693. Total and true digestible P contents of three MBM samples (MBM-1, MBM-2 and MBM-3) were determined to be 37.5 and 26.0; 60.2 and 36.6; and 59.8 and 25.1 g/kg, as fed basis, respectively, suggesting that P in MBM is not highly digestible. The overall data suggested that the use of regression approach to estimate true ileal P digestibility in feed ingredients has number of limitations. Overestimation as a result of using Ca- and P-deficient diets and the negative endogenous P losses observed for some ingredients (canola meal, sorghum and MBM-3) were main concerns. Negative ileal endogenous P losses were also shown to be associated with low true ileal P digestibility in these ingredients. In the final experiment (Chapter 8), two regression-based methodologies were compared for the measurement of true ileal P digestibility in maize and soybean meal. The results showed that the methodology influenced P digestibility in maize and soybean meal. The use of assay diets containing a narrow Ca:total P ratio yielded higher P digestibility for both ingredients. In this thesis research, the regression method was used to determine true ileal P digestibility of ingredients, but this approach suffers from several drawbacks. The data reported in this thesis also demonstrated that high dietary Ca concentrations were detrimental to the digestibility of nutrients, particularly of P, nitrogen and fat