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The interaction of hepatic lipase (HL) with heparan sulfate is critical to the function of this enzyme. The primary amino acid sequence of HL was compared to that of lipoprotein lipase (LPL), a related enzyme that possesses several putative heparin-binding domains. Of the three putative heparin-binding clusters of LPL (J. Biol. Chem. 1994. 269: 4626–4633; J. Lipid Res. 1998. 39: 1310–1315), one was conserved in HL (Cluster 1; residues Lys 297–Arg 300 in rat HL) and two were partially conserved (Cluster 2; residues Asp 307–Phe 320, and Cluster 4; residues Lys 337, and Thr 432–Arg 443). Mutants of HL were generated in which potential heparin-binding residues within Clusters 1 and 4 were changed to Asn. Two chimeras in which the LPL heparin-binding sequences of Clusters 2 and 4 were substituted for the analogous HL sequences were also constructed. These mutants were expressed in Chinese hamster ovary (CHO) cells and assayed for heparin-binding ability using heparin-Sepharose chromatography and a CHO cell-binding assay. The results suggest that residues within the homologous Cluster 1 region (Lys 297, Lys 298, and Arg 300), as well as some residues in the partially conserved Cluster 4 region (Lys 337, Lys 436, and Arg 443), are involved in the heparin binding of hepatic lipase. In the cell-binding assay, heparan sulfate-binding affinity equal to that of LPL was seen for the RHL chimera mutant that possessed the Cluster 4 sequence of LPL. Mutation of Cluster 1 residues of HL resulted in a major reduction in heparin binding ability as seen in both the cell-binding assay and the heparin-Sepharose elution profile. These results suggest that Cluster 1, the N-terminal heparin-binding domain, is of primary significance in RHL. This is different for LPL: mutations in the C-terminal binding domain (Cluster 4) cause a more significant shift in the salt required for elution from heparin-Sepharose than mutations in the N-terminal domain (Cluster 1). —Sendak, R. A., D. E. Berryman, G. Gellman, K. Melford, and A. Bensadoun. Binding of hepatic lipase to heparin: identification of specific heparin-binding residues in two distinct positive charge clusters.

The interaction of lipoprotein lipase (LPL) with heparan sulfate proteoglycans plays an important role in the metabolism and catalytic function of the enzyme. We have used site-directed mutagenesis to replace the basic residues contained in a discontinuous charge cluster (residues Lys 321, Arg 405, Arg 407, Lys 409, Lys 415, and Lys 416) of avian LPL with asparagine. The mutant proteins were expressed in Chinese hamster ovary cells and their affinity for heparin was evaluated by heparin-Sepharose chromatography. Mutation of residues Lys 321, Arg 405, Arg 407, Lys 409, and Lys 416 resulted in a decrease in affinity for heparin. The triple mutant LPL(R405N, R407N, K409N) possessed almost no high-affinity binding. The LPL mutants showed enzymatic activities ranging between 50–100% of that seen for wild-type LPL demonstrating that the overall structure of the enzyme was not significantly altered by the mutations. Mutation of previously identified heparin-binding regions of LPL results in a relatively small decrease in heparin-binding affinity, as compared with mutations in this carboxyl-terminal region, indicating that Lys 321, Arg 405, Arg 407, Lys 409, and Lys 416 constitute the major heparin-binding domain in LPL.—Sendak, R. A., and A. Bensadoun. Identification of a heparin-binding domain in the distal carboxyl-terminal region of lipoprotein lipase by site-directed mutagenesis. J. Lipid Res. 1998. 39: 1310–1315.

A panel of 13 monoclonal antibodies to avian lipoprotein lipase (LPL) was screened for inhibition of LPL binding to primary avian adipocytes. One monoclonal antibody, designated xCAL (monoclonal antibody to chicken adipose lipoprotein lipase) 3-6a, was found to inhibit the binding of LPL to primary avian adipocytes. In solid phase assays, xCAL 3-6a inhibited the binding of LPL to both heparan sulfate and heparin. XCAL 3-6a did not inhibit the catalytic activity of the avian enzyme. The monoclonal antibody was not found to cross-react significantly with bovine lipoprotein lipase. In order to determine the location of the epitope of xCAL 3-6a on lipoprotein lipase, several avian lipoprotein lipase deletion mutants were constructed and produced as glutathione S-transferase (GST) fusion proteins in E. coli. These mutants were screened for their ability to react with xCAL 3-6a using Western blotting. The minimum continuous fragment of lipoprotein lipase that was required for reactivity contained the amino acids 310 to 450. Site-directed mutagenesis of basic residues 321, 405, 407, 409, 415, and 416 revealed that Arg 405 is necessary for the interaction of LPL with xCAL 3-6a. Additional deletions of either the amino- or carboxyl-terminal portion of the fragment containing residues 310–450 resulted in loss of antibody binding, suggesting that the epitope is a discontinuous one that is formed when the termini are brought together through protein folding. Heparin-Sepharose chromatography of wild-type LPL and a mutant LPL in which the well-characterized heparin-binding sequence (Arg 281–Lys 282–Arg 284) has been mutated was carried out in the presence and absence of xCAL 3-6a. These experiments indicate that lipoprotein lipase contains a heparin-binding domain, in addition to Arg 281–Arg 284, that can be blocked by xCAL 3-6a.—Sendak, R. A., K. Melford, A. Kao, and A. Bensadoun. Identification of the epitope of a monoclonal antibody that inhibits heparin binding of lipoprotein lipase: new evidence for a carboxyl-terminal heparin-binding domain.

Background: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. Findings: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. Conclusions: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays. [ABSTRACT FROM AUTHOR]

A growth factor capable of stimulating DNA synthesis of Balb/c 3T3 cells was purified by heparin-Sepharose column chromatography about 1900-fold from the cytosol of human prostatic tissues obtained at autopsy or open prostatectomy. This growth factor bound to heparin-Sepharose in the presence of 0.5 mol/l NaCl and was eluted by 1.0-1.55 mol/l NaCl. Its molecular weight was estimated to be 68000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition was determined and compared with the data of other growth factors, which revealed no striking conformity. Distribution of growth factor activity was investigated in mechanically separated prostatic tissues of benign prostatic hyperplasia. The separation scheme provided two fractions: the stromal fraction consisting mainly of fibroblasts, fibers and smooth muscle, and the epithelial fraction consisting of epithelial cells. The specific growth-stimulating activity in the stromal fraction was about 2-fold that in the epithelial fraction. Referred to the total activity of whole tissue, about 74% of the activity could be detected in the stromal fraction, while only about 5% was detectable in the epithelial fraction. This study demonstrates the existence of a growth factor in human benign hyperplastic prostatic tissues, showing a remarkable distribution of growth factor activity, which may play a role in the pathogenesis of benign prostatic hyperplasia. [ABSTRACT FROM AUTHOR]