No abstract

The cerebral deposition of amyloid ␤-peptide is an early and critical feature of Alzheimer's disease. Amyloid ␤-peptide is released from the amyloid precursor protein by the sequential action of two proteases, ␤-secretase and ␥-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of ␤-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a se- , and Cys 330 -Cys 380 ). Despite the conservation of the active site residues and the 30 -37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member.The hallmarks of Alzheimer's disease (AD) 1 pathology are brain plaques and vascular deposits (1) consisting of the 4-kDa amyloid ␤-peptide (A␤) (2). Overproduction of the 42-amino acid form of A␤, A␤42, has been suggested to be the cause of all known cases of familial early onset AD (3), and it is assumed that A␤42 deposition plays an early and critical role in sporadic AD as well. Therefore, A␤ metabolism has attracted considerable interest. In 1987 it was shown (4) that formation of A␤ requires proteolytic cleavage of a large type I transmembrane protein, the ␤-amyloid precursor protein (APP), which is constitutively expressed in most cell types. Over the next decade the proteolytic processing of APP has been studied in great detail in a variety of systems by many groups. Taken together, these studies have shown that A␤ is generated at a low rate by most cells analyzed and that two different proteolytic activities are required for A␤ generation. First, ␤-secretase cleaves APP to generate the N terminus of A␤, and second, ␥-secretase cleaves the C terminus, leading to the release of A␤ (for review see Ref. 5). Studies with intact cells expressing APP and the endogenous secretases have led to conclusions about the properties of the ␤-and ␥-secretases, e.g. their tissue distribution, subcellular localization, substrate requirements (see e.g. Ref. 6) etc., but until recently the identity of both ␤-and ␥-secretase was unknown. This changed when we very recently identified the novel transmembrane aspartic protease BACE as the major ␤-secretase (7). Three subsequently published independent studies (8 -10) have confirmed this conclusion. Here we characterize the BACE protein. We show that BACE is an Nglycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We determine the processing and N-glycosylation sites and the disulfide bonds. Our results demonstrate that BACE is an unusual member of the pepsin family. EXPERIMENTAL PROCEDURESMat...

The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than fulllength huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeatcontaining huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

The leptin receptor (OB-R) is a member of the class I cytokine receptor family and mediates the weight regulatory effects of its ligand through interaction with cytoplasmic kinases. The extracellular domain of this receptor is comprised of two immunoglobulin-like and cytokine-receptor homology domains each and type III fibronectin domains. The extracellular domain of human leptin receptor was expressed in and purified from Chinese hamster ovary cells and was found to contain extensive N-glycosylation (approximately 36% of the total protein). The purified protein had a molecular weight of approximately 145,000 and exhibited ligand binding ability as evidenced by formation of ligand-receptor complex, followed by chemical cross-linking. The determined disulfide motif of the soluble leptin receptor contained several distinct cystine knots as well as 10 free cysteines. The N-glycosylation analysis revealed that Asn 624 of the WSXWS motif (residues 622-626) within the C-terminal cytokine receptor homology domain was glycosylated, indicating that this region is solvent-exposed. On the other hand, the N-terminal WSXWS motif was not glycosylated.The ob gene product, leptin, is an important circulating signal for regulation of body weight (1-4). Weight reducing effects of recombinant leptin were observed in both normal mice and mice with diet-induced obesity (5). The biological function of leptin is mediated through its membrane-associated receptor, leptin receptor (OB-R).1 Because of the biological and therapeutic importance of the leptin/OB-R system, numerous studies have been carried out to understand how this interaction is related to body weight regulation (6 -9).Human OB-R is a membrane-spanning glycoprotein consisting of a signal sequence, two immunoglobulin domains, two cytokine receptor homology (CRH) domains each containing a WSXWS motif, fibronectin type III domains, a transmembrane region, and an intracellular domain. The predicted extracellular domain consists of 839 amino acid residues and shows significant similarity to members of the class I cytokine receptor family (6), in particular the gp-130 signal-transducing component of various cytokine receptors as well as the granulocytecolony-stimulating factor receptor (G-CSFR) (10). Although overall sequence identity between OB-R and gp-130 is low (approximately 24%), several key regions are conserved, particularly the second cytokine homology domain (CRH)-2 (6). In this paper, we report structural information including the disulfide motif and N-glycosylation map of the extracellular domain of OB-R. EXPERIMENTAL PROCEDURESMaterials-Pepsin was obtained from Sigma. Trypsin and thermolysin were purchased from Boehringer Mannheim. Cross-linkers BS and DSS were obtained from Pierce and 4-HCCA from Sigma. The N-and O-glycanases were purchased from Genzyme (Cambridge, MA), and sialidase was from Boehringer Mannheim. Recombinant human leptin was prepared as described previously (11). All chemicals used were of reagent grade or analytical grade.Antisera Preparation-New ...

We have assigned the disulfide structure of Md-65 agouti-related protein (Md65-AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coil cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65-AGRP were partially reduced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N-ethylmaleimide (NEM). The procedure generated several isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide bonds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37, 12172-12177].

Glial cell line-derived neurotrophic factor is a protein known to enhance the survival of dopaminergic neurons against several neurotoxins. It has been shown to have therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. We have determined the inter- and intramolecular disulfide linkages of the dimeric molecule by a combination of direct peptide analysis and peptide analysis after either partial reduction or partial oxidation of the protein. Under an acidic condition, the interchain disulfide bond was selectively cleaved with tris(2-carboxyethyl)phosphine, revealing that Cys101 was involved in the intermolecular disulfide linkage. Three other disulfides, Cys68-Cys131, Cys72-Cys133, and Cys41-Cys102, were identified as intramolecular linkages. The determined disulfide structure is highly homologous to that of transforming growth factor beta 2. Since one intramolecular disulfide points through a ring consisting of eight amino acid residues based on the similarity with transforming growth factor beta 2, the disulfide-linked peptides were not purified by conventional methods. Only the peptides from an N-terminal region (residues -1 to 37) were liberated by proteolytic treatment with trypsin or endoproteinase Lys-C, resulting in a stable cystine-knot protein.

Estrogens have been suggested to exhibit neuroprotective activities against several insults including beta-amyloid and glutamate, one of the excitatory neurotransmitters in the central nervous system. In the present study, we showed that exposure to glutamate not only inhibited the cell growth of exponentially growing rat pheochromocytoma PC12 cells in a time- and dose-dependent manner, but also influenced cell adherence capacity. Glutamate-induced growth inhibition was significantly attenuated by the co-administration of estradiol in PC12 cells. Pre-exposure of the PC12 cells to the estradiol was not required for protection against glutamate-induced growth inhibition. Administration of anti-estrogen ICI182,780 efficiently blocked the neuroprotective effects of estradiol. Glutamate-induced changes in cell adherence, on the other hand, were not significantly affected by estradiol. These data indicate that the neuroprotective effects of estradiol against glutamate-induced insults in PC12 cells, at least in part, involve estrogen receptor-dependent pathways.