The integrins ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 are referred to as a collagen receptor subgroup of the integrin family. Recently, both ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O ‫؍‬ hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, ␣ 11 ␤ 1 , recognizes collagens using C2C12 cells transfected with ␣ 11 cDNA and the bacterially expressed recombinant ␣ 11 I domain. The ligand binding properties of ␣ 11 ␤ 1 were compared with those of ␣ 2 ␤ 1 . Mg 2؉ -dependent ␣ 11 ␤ 1 binding to type I collagen required micromolar Ca 2؉ but was inhibited by 1 mM Ca 2؉ , whereas ␣ 2 ␤ 1 -mediated binding was refractory to millimolar concentrations of Ca 2؉ . The bacterially expressed recombinant ␣ 11 I domain preference for fibrillar collagens over collagens IV and VI was the same as the ␣ 2 I domain. Despite the difference in Ca 2؉ sensitivity, ␣ 11 ␤ 1 -expressing cells and the ␣ 11 I domain bound to helical GFOGER sequences in a manner similar to ␣ 2 ␤ 1 -expressing cells and the ␣ 2 I domain. Modeling of the ␣ I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , and also ␣ 11 ␤ 1 integrins. Although ␣ 10 and ␣ 11 chains show the highest sequence identity, ␣ 2 and ␣ 11 are more similar with regard to collagen specificity. Future studies will reveal whether ␣ 2 ␤ 1 and ␣ 11 ␤ 1 integrins also show overlapping biological functions.The collagen family currently includes at least 24 members (1, 2), and four different collagen-binding integrins ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 (3) and ␣ 11 ␤ 1 (4) are known. The ␣ 3 ␤ 1 integrin does not interact directly with collagen, but it does act as a laminin receptor (5) that can affect the activity of the collagen receptor ␣ 2 ␤ 1 through receptor cross-talk (6).

No abstract

3040 Poster Board II-1016 The physiologic function of factor XII (XII) is not known. New interest in XII has occurred because XII KO mice are protected from thrombosis. Exposed arterial collagen, aggregated protein, RNA, and platelet polysomes are recently recognized entities in developing thrombus that promote XII autoactivation to XIIa increasing thrombus formation, independent of a role in hemostasis. However, these observations do not indicate a constitutive, physiologic function for XII. We sought a physiologic function for zymogen XII. XII has been recognized to stimulate MAP kinase. Cleaved high molecular weight kininogen (HKa) is known to be antiangiogenic. Further, XII and HK mutually block each others binding to endothelial cells. Both XII and HKa bind urokinase plasminogen activator receptor (uPAR) at an overlapping site. We investigated if XII stimulates cells by interacting with uPAR and if this activity influences angiogenesis. XII (3-200 nM) with 0.05 mM Zn ion induces ERK1/2 (MAPK44 and 42) and Akt (Ser473) phosphorylation in endothelial cells. XII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. The uPAR signaling region for XII is on domain 2 adjacent to uPAR's integrin binding site. HKa or peptides from HKa's domain 5 inhibit XII-induced ERK1/2 and Akt phosphorylation. A beta-1-integrin peptide that binds uPAR, antibody 6S6 to beta-1-integrin, or the EGFR inhibitor AG1478 blocks XII-induced phosphorylation of ERK1/2 and Akt. XII induces endothelial cell proliferation and 5-bromo-2'deoxy-uridine incorporation. XII stimulates aortic sprouting in normal but not uPAR deficient mouse aorta and this mechanism is blocked by PD98059, LY294002, AG1478, or HKa. XII also induces angiogenesis in matrigel plugs. Finally, XII knockout mice have reduced constitutive and wound-induced blood vessel number on initial biopsy and 9 days after wound healing, respectively. In sum, XII initiates outside-in signaling mediated by uPAR, beta-1-integrin, and the EGFR leading to HUVEC proliferation, growth, and angiogenesis. XII is a constitutive proangiogenic protein. Disclosures No relevant conflicts of interest to declare.

High fructose consumption in the Western diet correlates with disease states such as obesity and metabolic syndrome complications, including type II diabetes, chronic kidney disease, and nonalcoholic fatty acid liver disease. Liver and kidneys are responsible for metabolism of 40–60% of ingested fructose, while the physiological fate of the remaining fructose remains poorly understood. The primary metabolic pathway for fructose includes the fructose-transporting solute-like carrier transport proteins 2a (SLC2a or GLUT), including GLUT5 and GLUT9, ketohexokinase (KHK), and aldolase. Bioinformatic analysis of gene expression encoding these proteins (glut5, glut9, khk, and aldoC, respectively) identifies other organs capable of this fructose metabolism. This analysis predicts brain, lymphoreticular tissue, placenta, and reproductive tissues as possible additional organs for fructose metabolism. While expression of these genes is highest in liver, the brain is predicted to have expression levels of these genes similar to kidney. RNA in situ hybridization of coronal slices of adult mouse brains validate the in silico expression of glut5, glut9, khk, and aldoC, and show expression across many regions of the brain, with the most notable expression in the cerebellum, hippocampus, cortex, and olfactory bulb. Dissected samples of these brain regions show KHK and aldolase enzyme activity 5–10 times the concentration of that in liver. Furthermore, rates of fructose oxidation in these brain regions are 15–150 times that of liver slices, confirming the bioinformatics prediction and in situ hybridization data. This suggests that previously unappreciated regions across the brain can use fructose, in addition to glucose, for energy production.

Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable Aspergillus fumigatus phytase (Afp), suggest associations of thermostability with several key residues (E35, S42, R168, and R248) that form a hydrogen bond network in the E35-to-S42 region and ionic interactions between R168 and D161 and between R248 and D244. In this study, loss-of-function mutations (E35A, R168A, and R248A) were introduced singularly or in combination into seven mutants of Afp. All seven mutants displayed decreases in thermostability, with the highest loss (25% [P < 0.05]) in the triple mutant (E35A R168A R248A). Subsequently, a set of corresponding substitutions were introduced into nine mutants of PhyA to strengthen the hydrogen bonding and ionic interactions. While four mutants showed improved thermostability, the best response came from the quadruple mutant (A58E P65S Q191R T271R), which retained 20% greater (P < 0.05) activity after being heated at 80°C for 10 min and had a 7°C higher melting temperature than that of wild-type PhyA. This study demonstrates the functional importance of the hydrogen bond network and ionic interaction in supporting the high thermostability of Afp and the feasibility of adopting these structural units to improve the thermostability of a homologous PhyA phytase.Phytase catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate), the major storage form of phosphorus in plant seeds (20), to phosphate and myo-inositol. The enzyme has been effectively used as an animal feed supplement to improve the bioavailability of phytate phosphorus to simplestomached swine and poultry (4, 13). Because temperatures in the practical feed pelleting process can reach as high as 70 to 90°C (18), phytase enzymes with sufficiently high thermal stability are desirable but rare among the naturally occurring sources (14). Therefore, different strategies have been used to enhance the thermal stability of phytase (22).Aspergillus fumigatus phytase (Afp) (19) is a well-known heat-resilient phytase; it retains 90% of its initial activity after being heated at 100°C for 20 min (21). In comparison, Aspergillus niger PhyA phytase (30) displays much less heat resistance, despite a higher specific activity and a better pH profile than those of Afp (17,29,33,34). More intriguingly, these two phytases have 66% sequence homology and very similar overall crystal structures (9,15,35). Both enzymes contain a small ␣ domain and a large ␣/␤ domain. The small ␣ domain is composed of a long ␣ helix and seven short ␣ helices, and the large ␣/␤ domain contains a six-stranded ␤ sheet surrounded by two long ␣ helices at one side and several short ␣ helices at the other side. Nevertheless, detailed structure comparisons between these two enzymes indicate that three amino acid residues in Afp-E35, R168, and R248-may be critical...

alpha 2-macroglobulin is the major inhibitor of PSA when it reached the circulation. Contrary to earlier assumptions, nicked PSA can bind to A2M rendering it inaccessible to antibodies.

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M r of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.

No abstract