Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

COMPARATIVE MORPHOLOGY OF THE PRIMARY VASCULAR SYSTEMS IN SOME SPECIES OF ROSACEAE AND LEGUMINOSAE

The structural patterns of the primary vascular systems in some species of Leguminosae and Rosaceae have been determined by tracing the longitudinal course of the vascular bundles in terminal stem segments. These systems are interpreted as consisting of sympodia. Each sympodium is composed of an axial bundle which is continuous through the length of the segment and from which arise trace bundles that supply leaves and axillary buds. A compact arrangement of vascular bundles seems to correlate with the woody habit. Regardless of the degree of compactness of the primary vascular system, the structural identity of the individual sympodia is maintained. The total number of vascular bundles at a particular level is related to the number of axial bundles in the system, the number of traces per leaf and per axillary bud, and the number of internodes traversed by the traces prior to entering a lateral appendage. Shrubs and trees have more vascular bundles than herbs. Data from this study and the literature indicate that the vascular system is predominantly of the open type in dicotyledonous plants which have helically arranged leaves and, further, that in such plants with a 3-trace, trilacunar nodal structure, the number of sympodia coincides with the number of orthostichies (which is also the denominator of the phyllotactic fraction). In open systems leaf gaps cannot be morphologically delimited. Because of the resemblance of the open type of angiosperm vascular system to that of certain gymnosperms, previously interpreted to have evolved from a protostele, we suggest that the eustele of angiosperms is homologous with the stele of gymnosperms. We believe, also, that angiosperms, like gymnosperms, are probably not characterized by leaf gaps of filicinean type. We provide, furthermore, a rationale for the view that the axial bundle of a sympodium is a cauline structure.     

Disruption of alpha-mannosidase processing induces non-canonical hybrid-type glycosylation

Golgi alpha-mannosidase II is essential for the efficient formation of complex-type glycosylation. Here, we demonstrate that the disruption of Golgi alpha-mannosidase II activity by swainsonine in human embryonic kidney cells is capable of inducing a novel class of hybrid-type glycosylation containing a partially processed mannose moiety. The discovery of 'Man(6)-based' hybrid-type glycans reveals a broader in vivo specificity of N-acetylglucosaminyltransferase I, further defines the arm-specific tolerance of core alpha1-6 fucosyltransferase to terminal alpha1-2 mannose residues, and suggests that disruption of Golgi alpha-mannosidase II activity is capable of inducing potentially 'non-self' structures.     

c-Jun Is critical for the progression of osteosarcoma: proof in an orthotopic spontaneously metastasizing model

The oncogene c-Jun has been found to be up-regulated in a variety of cancers including osteosarcoma. DNA enzymes (DNAzymes) are oligonucleotides capable of specific catalysis of target mRNA. A c-Jun DNAzyme inhibited the growth and metastasis of osteosarcoma in an orthotopic spontaneously metastasizing model of the disease. c-Jun down-regulation-mediated apoptosis in osteosarcoma cells involved caspase-1, caspase-2, and caspase-8, but not the Fas/FasL pathway. Clinically, knockdown of c-Jun with DNAzymes may proffer an improved treatment outcome for these tumors originating in bone.     

Selective deactivation of serum IgG: a general strategy for the enhancement of monoclonal antibody receptor interactions

Serum IgG is a potent inhibitor of monoclonal antibody (mAb) binding to the cell-surface Fcγ receptors (FcγRs), which mediate cytotoxic and phagocytic effector functions. Here, we show that this competition can be eliminated, selectively, by the introduction to serum of (i) an enzyme that displaces Fc from FcγRs and (ii) a modification present in the therapeutic mAb that renders it resistant to that enzyme. Specifically, we show that (i) EndoS (endoglycosidase S) cleaves only complex-type glycans of the type found on IgG but (ii) is inactive against an engineered IgG Fc with oligomannose-type glycans. EndoS thus reduces FcγR binding of serum IgG, but not that of engineered mAb. Introduction of both the engineered mAb and endoglycosidase in serum leads to a dramatic increase in FcγR binding compared to the introduction of mAb in serum alone. Antibody receptor refocusing is a general technique for boosting the effector signal of therapeutic antibodies.