Alzheimer disease is intimately linked to an excess amount of amyloid-beta (Abeta) in the brain. Thus, therapeutic inhibition of Abeta production is an attractive clinical approach to treat this disease. Here we provide the first direct experimental evidence that the treatment of Tg2576 transgenic mice with an inhibitor of beta-secretase, GRL-8234, rescues the age-related cognitive decline. We demonstrated that the injected GRL-8234 effectively enters the brain and rapidly decreases soluble Abeta in the brain of Tg2576 mice. The rescue of cognition, which was observed only after long-term inhibitor treatment ranging from 5 to 7.5 mo, was associated with a decrease of brain amyloid-beta plaque load. We also found no accumulation of amyloid-beta precursor protein after several months of inhibitor treatment. These observations substantiate the idea that Abeta accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of Abeta reduction therapy as a treatment of AD.
Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1-538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 A resolution. The crystal form belongs to space group P2(1)2(1)2(1) with one monomer per asymmetric unit, and the protein shows an alpha/beta hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115-125 and 270-285).
Title: Proline-rich domain and glycosylation are not essential for the enzymic activity of bile salt-activated lipase. Kinetic studies of T-BAL, a truncated form of the enzyme, expressed in Escherichia coli Downs D, Xu YY, Tang J, Wang CS Ref: Biochemistry, 33:7979, 1994 : PubMed
We have expressed and purified a truncated recombinant human milk bile salt-activated lipase (T-BAL) from the T7 expression system in Escherichia coli. This T-BAL contains the N-terminal 538 residues of the 722-residue native enzyme. The purified T-BAL, when assayed with PANA (p-nitrophenyl acetate), had a specific activity of 64 +/- 2 units/mg (n = 4), as compared to 52 units/mg for the native enzyme. Because the recombinant T-BAL expressed in E. coli is not glycosylated, these results indicated that the highly glycosylated C-terminal region of BAL is not essential for catalytic function. Heat inactivation patterns of native BAL and T-BAL were found to be similar, further suggesting that the folding of T-BAL is similar to that of the catalytic domain of the native enzyme. With the availability of a sufficient amount of recombinant T-BAL, the specificity and kinetics of T-BAL and native BAL were compared. Fluorescence studies of T-BAL indicated that it has a slightly higher affinity for the monomeric form of taurocholate with a dissociation constant (KA) of 0.32 mM, compared with the reported 0.37 mM for the native enzyme. Further kinetic analysis indicated that there are enzyme specificity changes revealed with the use of PANA and PANB (p-nitrophenyl butyrate) as substrates. When assayed in the presence of taurocholate, T-BAL has a higher turnover rate constant with p-nitrophenyl acetate than with p-nitrophenyl butyrate, which was found to be in contrast to native BAL..
The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in lambda gt11 and lambda gt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDNA as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. The deduced BAL protein structure contains in the carboxyl-terminal region 16 repeating units of 11 amino acids each. The repeating units have the basic structure Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro with only minor substitutions. The amino acid sequence of human BAL is related to that of pancreatic lysophospholipase, cholesterol esterase, cholinesterase, acetylcholinesterase, and thyroglobulin. Ten of the 14 cyanogen bromide fragments of diisopropyl fluorophosphate inhibited human milk BAL were isolated, determined for N-terminal sequences, analyzed for amino sugars, and tested for some functional properties. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. We have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL.
The fatty acid specificity of the bile salt-activated lipase purified from human milk was studied using C12 to C54 (total acyl carbon) saturated and the C54 unsaturated triacylglycerols. Kinetic studies indicated that the short chain triacylglycerols were hydrolyzed more readily than the long chain triacylglycerols, and that the long chain unsaturated triacylglycerols were attacked more readily than the long chain saturated triacylglycerols. This fatty acid specificity was also apparent intramolecularly, both short chain and unsaturated fatty acids being released at higher rates than the saturated long chain acids. The enzyme possessed neither positional specificity nor stereospecificity as indicated by the nearly simultaneous appearance of the sn-1,2-, sn-2,3-, and sn-1,3-dioleoylglycerols from trioleoylglycerol. The hydrolyses of these three ester bonds were approximately at their anticipated chemical reactivities. Synthetic rac-1-monooleoylglycerols were hydrolyzed about 2 times faster than the sn-2-monooleoylglycerols. It is concluded that the bile salt-activated lipase may possess a special potential for a rapid release of short chain and polyunsaturated fatty acids from dietary triacylglycerols in the intestinal lumen of infants.
