Characterization of the high affinity heparin binding site of the Alzheimer's disease βA4 amyloid precursor protein (APP) and its enhancement by zinc(II)

The Alzheimer's disease βA4 amyloid precursor protein (APP) has been shown to be involved in a diverse set of biological protein precursor-like proteins (APLP1 and APLP2) belong to a superfamily of proteins that are probably functionally related. In order to characterize the cell adhesion properties of APP the brain specific isoform APP₆₉₅ was purified and used to assess the binding to herparin, a structural and functional analogue of the glycosaminoglycan heparan sulfate. We show that APP binds in a time dependent and saturable manner to heparin. The salt concentration of 620 mM at which APP elutes from heparin Sepharose is greater than physiological. Tha apparent equilibrium constant for dissociation was determined to be 300 pM for APP binding to heparin Sepharose. A high affinity heparin binding site was identified within a region conversed in rodent and human APP, APLP1 and APLP2. This binding site was located between residues 316-337 of APP₆₉₅ which is within the carbohydrate domain of APP. We also demonstrate an interaction between this heparin binding site and the zinc(II) binding site which is conserved in all members of the APP superfamily. We show by using an automated surface plasmon resonance biosensor (BIAcore, Pharmacia) that the affinity for heparin is increased two- to four-fold in the presence of micromolar zinc(II). The identification of zinc-enhanced binding of APP to heparin sulfate side chains of proteoglycans offers a molecular link between zinc(II), as a putative environmental toxin for Alzheimer's disease, and aggregation of amyloid βA4 protein.     

Heparin-inhibitable lectins: Marked similarities in chicken and rat

Extracts of young rat lung contain a heparin-inhibitable lectin that closely resembles one recently purified from chicken liver. Both lectins interact with heparin and N-acetyl-D-galactosamine, and were purified by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. They both behave as high molecular weight aggregates that can be dissociated into two peptides with apparent molecular weights of 13,000 and 16,000 by gel electrophoresis in SDS. Samples of purified lectin contained up to 20% DNA by weight, and the degree of lectin aggregation and hemagglutination activity was greatly reduced by treatment with micrococcal nuclease without inhibiting heparin-binding activity. Association of lectin with DNA is an artifact of homogenization in high salt, since only 2% of the lectin is found associated with a purified nuclear fraction.     

Endogenous lectins in chickens and slime molds: Transfer from intracellular to extracellular sites

Endogenous lectins in both cellular slime molds and chicken tissues have been localized primarily intracellularly, in contrast with the predominantly extracellular localization of the glycoproteins, glycolipids, and glycosaminoglycans with which they might interact. Here we present evidence that lectins in both of these organisms may be externalized and become associated with the cell surface and/or extracellular materials. In chicken intestine, chicken-lactose-lectin-II is shown to be localized in the secretory granules of the goblet cells, along with mucin, and to be secreted onto the intestinal surface. In embryonic muscle, chicken-lactose-lectin-I is shown to be externalized with differentiation, ultimately becoming localized on the surface of myotubes and in the extracellular spaces. In a cellular slime mold, Dictyostelium purpureum, externalization of lectin is elicited by either polyvalent glycoproteins that bind the small amount of endogenous cell surface lectin, or by slime mold or plant lectins that bind unoccupied complementary cell surface oligosaccharides. These results suggest that externalization of endogenous lectin may be a response to specific external signals. We conclude that lectins are frequently held in intracellular reserves awaiting release for specific external functions.     

Detection and Classification of Trypanosoma cruzi by DNA Hybridization with Nonradioactive Probes

ABSTRACT. Total or kinetoplast DNA (kDNA) from 72 isolates and clones of Trypanosoma cruzi as well as from nine related trypanosomatids were analyzed by dot hybridization using nonradioactive kDNA or cloned minicircle fragments as probes. Biotinylated-kDNA probes generated by nick-translation proved reliable for distinguishing Zymodeme 1 and Zymodeme 2bol of T. cruzi parasites. In contrast, digoxigenin-labeled kDNA obtained by random-priming did not distinguish among T. cruzi isolates but did distinguish among New World leishmanias. Cloned minicircle fragments labeled with digoxigenin gave the same results as digoxigenin-labeled kDNA, except for a 10-fold decrease in sensitivity. Digoxigenin-labeled DNA probes proved useful in unambiguously detecting T. cruzi from different geographic regions of America. However, T. rangeli and T. cruzi marinkellei were not distinguished by these probes.     

Thermosensitive heparin‐poloxamer hydrogel encapsulated bFGF and NGF to treat spinal cord injury

The application of growth factors (GFs) for treating chronic spinal cord injury (SCI) has been shown to promote axonal regeneration and functional recovery. However, direct administration of GFs is limited by their rapid degradation and dilution at the injured sites. Moreover, SCI recovery is a multifactorial process that requires multiple GFs to participate in tissue regeneration. Based on these facts, controlled delivery of multiple growth factors (GFs) to lesion areas is becoming an attractive strategy for repairing SCI. Presently, we developed a GFs‐based delivery system (called GFs‐HP) that consisted of basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and heparin‐poloxamer (HP) hydrogel through self‐assembly mode. This GFs‐HP was a kind of thermosensitive hydrogel that was suitable for orthotopic administration in vivo. Meanwhile, a 3D porous structure of this hydrogel is commonly used to load large amounts of GFs. After single injection of GFs‐HP into the lesioned spinal cord, the sustained release of NGF and bFGF from HP could significantly improve neuronal survival, axon regeneration, reactive astrogliosis suppression and locomotor recovery, when compared with the treatment of free GFs or HP. Moreover, we also revealed that these neuroprotective and neuroregenerative effects of GFs‐HP were likely through activating the phosphatidylinositol 3 kinase and protein kinase B (PI3K/Akt) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signalling pathways. Overall, our work will provide an effective therapeutic strategy for SCI repair.     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.     

Heparin-functionalized thermoresponsive surface

Temperature-dependent regulation of affinity binding between bioactive ligands and their cell membrane receptors is an attractive approach for the dynamic control of cellular adhesion, proliferation, migration, differentiation, and signal transduction. Covalent conjugation of bioactive ligands onto thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces facilitates the modulation of one-on-one affinity binding between bioactive ligands and cellular receptors by changing temperature. For the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins, thermoresponsive cell culture surface modified with heparin, which interacts with heparin-binding proteins such as basic fibroblast growth factor (bFGF), has been proposed. Heparin-functionalized thermoresponsive cell culture surface induces (1) the multivalent affinity binding of bFGF in active form and (2) accelerating cell sheet formation in the state of shrunken PIPAAm chains at 37°C. By lowering temperature to 20°C, the affinity binding between bFGF and immobilized heparin is reduced with increasing the mobility of heparin and the swollen PIPAAm chains, leading to the detachment of cultured cells. Therefore, heparin-functionalized thermoresponsive cell culture surface was able to enhance cell proliferation and detach confluent cells as a contiguous cell sheet by changing temperature. A cell cultivation system using heparin-functionalized thermoresponsive cell culture surface is versatile for immobilizing other heparin-binding proteins such as vascular endothelial growth factor, fibronectin, antithrombin III, and hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species.