Seokjoo Hong

Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln29–His53, which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu32–His55. We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3-/- mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.

Complementing Substitutions at the Bottom of the Barrel Influence Catalysis and Stability of Ribulose-bisphosphate Carboxylase/Oxygenase

The temperature-conditional photosynthesis-deficient mutant 68-4PP of Chlamydomonas reinhardtii results from a Leu-290 to Phe substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Although this substitution occurs relatively far from the active site, the mutant enzyme has a reduced ratio of carboxylation to oxygenation in addition to reduced thermal stability in vivo and in vitro. In an attempt to understand the role of this region in catalysis, photosynthesis-competent revertants were selected. Two revertants, named R96-4C and R96-8E, were found to arise from second-site mutations that cause V262L and A222T substitutions, respectively. These intragenic suppressor mutations increase the CO2/O2 specificity and carboxylation Vmax back to wild-type values. Based on the crystal structure of the spinach holoenzyme, Leu-290 is not in van der Waals contact with either Val-262 or Ala-222. However, all three residues are located at the bottom of the α/β-barrel active site and may interact with residues of the nuclear encoded small subunits. It appears that amino acid residues at the interface of large and small subunits can influence both stability and catalysis.

Thermal Instability of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from a Temperature-Conditional Chloroplast Mutant of Chlamydomonas reinhardtii

Mutant 68–4PP of Chlamydomonas reinhardtii has only 10% of the normal level of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme when grown at 35[deg]C. However, when grown at 25[deg]C, the amount of holoenzyme is greater than 35% of the wild-type level, and the purified enzyme has a reduced CO2/O2 specificity factor. These mutant characteristics result from a chloroplast mutation that causes leucine-290 to be replaced by phenylalanine within the Rubisco large-subunit protein. A nuclear mutation (named S52–2B) was previously identified that can suppress both the in vivo instability and reduced CO2/O2 specificity of the mutant enzyme. However, the effect of this nuclear mutation on the in vitro stability of the holoenzyme was not resolved. In the present study, purified Rubisco from mutant 68–4PP was found to be less thermally stable than the wild-type enzyme, and it had maximal carboxylase activity at a lower temperature. When incubated at 35[deg]C, the mutant enzyme lost carboxylase activity at a much faster rate than the wild-type enzyme. However, the nuclear S52–2B suppresor mutation improved the thermal stability of the mutant enzyme in all cases. These results indicate that structural changes in mutant 68–4PP Rubisco can account for its observed inactivation in vitro and degradation in vivo. Such structural alterations are alleviated by the function of a nuclear gene.

Nuclear Mutation Inhibits Expression of the Chloroplast Gene That Encodes the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Chlamydomonas reinhardtii mutant 76–5EN was recovered as a light-sensitive, acetate-requiring strain that failed to complement a chloroplast structural gene mutant of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Further genetic analysis revealed that the new mutation was inherited in a mendelian pattern, indicating that it resides within the nucleus. The 76–5EN mutant lacks Rubisco holoenzyme but has wild-type levels of whole-chain electron transport activity and chlorophyll. During a 1-min pulse labeling with 35SO42-, little or no Rubisco large-subunit synthesis occurred in the mutant. Nuclear-encoded small subunits were synthesized to a normal level and were subsequently degraded. When analyzed by northern hybridization, the 76–5EN mutant was found to have a decreased level of large-subunit mRNA. Large-subunit mRNA synthesis also appeared to be reduced during a 10-min pulse labeling with [32P]orthophosphate, but the labeled mRNA was stable during a 1-h chase. These results indicate that a nuclear gene mutation specifically disrupts the accumulation of large-subunit mRNA within the chloroplast. A deeper understanding of the nature of the 76–5EN gene may be useful for manipulating the expression of the agronomically important Rubisco enzyme.

Temperature-conditional nuclear mutation of Chlamydomonas reinhardtii decreases the CO2/O2 specificity of chloroplast ribulosebisphosphate carboxylase/oxygenase

The Chlamydomonas reinhardtii (Dangeard) temperature-conditional mutant 68-11AR is phenotypically indistinguishable from the wild type at the permissive temperature (25°C), but has greatly reduced photosynthetic ability and requires acetate for growth at the restrictive temperature (35°C). The mutant strain is deficient in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) holoenzyme when grown at 35°C. This decrease in the level of enzyme appears to be due to degradation of assembled holoenzyme rather than to a reduction in the synthesis of enzyme subunits. When grown at 25°C, the mutant has a substantial amount of Rubisco. Enzyme purified from 25°C-grown mutant cells was found to have a 16% decrease in the CO2/O2 specificity factor when compared to the wild-type enzyme. This alteration was accompanied by changes in the kinetic constants for both carboxylation and oxygenation. Although the Rubisco active site is located on the chloroplast-encoded large subunit, genetic analysis showed that the 68-11AR strain arose from a nucleargene mutation. The two nuclear genes that encode the Rubisco small subunits (rbcS1 and rbcS2) were cloned from mutant 68-11AR and completely sequenced, but no mutation was found. Analysis of restriction-fragment length polymorphisms also failed to detect linkage between mutant and rbcS gene loci. These results indicate that nuclear genes can influence Rubisco catalysis without necessarily encoding polypeptides that reside within the holoenzyme.