Role of the methoxy group in immune responses to mPEG-protein conjugates

Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD ("relative titer") had a median of 1.1 (range 0.9-1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1-20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.     

Novel pro-survival functions of the Kruppel-like transcription factor Egr2 in promotion of macrophage colony-stimulating factor-mediated osteoclast survival downstream of the MEK/ERK pathway

Determining the underlying mechanisms of macrophage colony-stimulating factor (M-CSF)-mediated osteoclast survival may be important in identifying novel approaches for treating excessive bone loss. This study investigates M-CSF-mediated MEK/ERK activation and identifies a downstream effector of this pathway. M-CSF activates MEK/ERK and induces MEK-dependent expression of the immediate early gene Egr2. Inhibition of either MEK1/2 or inhibition of Egr2 increases osteoclast apoptosis. In contrast, wild-type Egr2 or an Egr2 point mutant unable to bind the endogenous repressors Nab1/2 (caEgr2) suppresses basal osteoclast apoptosis and rescues osteoclasts from apoptosis induced by MEK1/2 or Egr2 inhibition. Mechanistically, Egr2 induces pro-survival Blc2 family member Mcl1 while stimulating proteasome-mediated degradation of pro-apoptotic Bim. In addition, Egr2 increased the expression of c-Cbl, the E3 ubiquitin ligase that catalyzes Bim ubiquitination. M-CSF, therefore, promotes osteoclast survival through MEK/ERK-dependent induction of Egr2 to control the Mcl1/Bim ratio, documenting a novel function of Egr2 in promoting survival.     

Loss of ERE binding activity by estrogen receptor-alpha alters basal and estrogen-stimulated bone-related gene expression by osteoblastic cells

Estrogen receptor (ER)-alpha can signal either via estrogen response element (ERE)-mediated pathways or via alternate pathways involving protein-protein or membrane signaling. We previously demonstrated that, as compared to wild type (WT) controls, mice expressing a mutant ER-alpha lacking the ability to bind EREs (non-classical estrogen receptor knock-in (NERKI)) display significant impairments in the skeletal response to estrogen. To elucidate the mechanism(s) underlying these in vivo deficits, we generated U2OS cells stably expressing either WT ER-alpha or the NERKI receptor. Compared to cells transfected with the control vector, stable expression of ER-alpha, even in the absence of E2, resulted in an increase in mRNA levels for alkaline phosphatase (AP, by 400%, P < 0.01) and a decrease in mRNA levels for insulin growth factor-I (IGF-I) (by 65%, P < 0.001), with no effects on collagen I (col I) or osteocalcin (OCN) mRNA levels. By contrast, stable expression of the NERKI receptor resulted in the suppression of mRNA levels for AP, col I, OCN, and IGF-I (by 62, 89, 60, and 70%, P < 0.001). While E2 increased mRNA levels of AP, OCN, col I, and IGF-I in ER-alpha cells, E2 effects in the NERKI cells on AP and OCN mRNA levels were attenuated, with a trend for E2 to inhibit col I mRNA levels. In addition, E2 had no effects on IGF-I mRNA levels in NERKI cells. Collectively, these findings indicate that ERE signaling plays a significant role in mediating effects of estrogen on osteoblastic differentiation markers and on IGF-I mRNA levels.     

The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse.

Heparan sulfate (HS) is a co-receptor for a number of growth factors, morphogens, and adhesion proteins. HS biosynthetic modifications may determine the strength and outcome of HS-ligand interactions. We previously described the phenotype of mice with a gene-trap mutation in Hs2st, encoding the key HS 2-O-sulfotransferase enzyme in HS polymer modification. In contrast to the early developmental failure of embryos lacking HS, the onset of abnormalities in the Hs2st(-/-) mice occurs only after midgestation, the most dramatic being the complete failure of kidney development. Uronate 2-O-sulfates were not detected in the mutant HS, indicating a complete loss of function of Hs2st. However, the domain structure of the mutant HS is conserved, and compensatory increases in N- and 6-O-sulfation maintain the overall charge density. The apparent affinities of the mutant HS for hepatocyte growth factor/scatter factor and fibronectin were unchanged but were reduced for fibroblast growth factor-1 and -2. Surprisingly, the Hs2st(-/-) cells were able to mount an apparently normal signaling response to fibroblast growth factor-1 and -2 as well as to hepatocyte growth factor/scatter factor.     

Monitoring endoplasmic reticulum-to-Golgi traffic of a plant calreticulin by protein glycosylation analysis

Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.     

Butterfly pollination and high-contrast visual signals in a low-density distylous plant

In low-density butterfly-pollinated Mussaenda frondosa (Rubiaceae), flowers attract pollinators at short distances while conspicuous, non-rewarding accessory bracts are detectable at long distances by long-ranging pollinators such as the birdwing butterfly Troides minos that did not detect flower-bearing plants in the absence of these bracts. However, even in the absence of flowers, the white, ultraviolet-absorbing bracts attracted butterflies that visited flowerless plants. Although flower visits by short-ranging territorial butterflies declined significantly on removal of bracts, they did not cease completely. Nectar-robbing carpenter bees and birds did not change their behaviour following bract removal. Bract removal caused a significant decline in fruit set, indicating their importance as visual signals to pollinators.     

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.