The influence of the noradrenergic/stress system on perceptual biases for reward.

Previous research has established a role for the norepinephrine (NE)/stress system in individual differences in biases to attend to reward or punishment. Outstanding questions concern its role in the flexibility with which such biases can be changed. The goal of this preregistered study was to examine the role of the NE/stress system in the degree to which biases can be trained along the axis of valence in the direction of reward. Participants genotyped for a common deletion variant of ADRA2b (linked to altered NE availability) experienced either an acute stress induction or a control procedure. Following stress induction, a "bias probe" task was presented before and after training. In the bias probe task, participants made forced choice judgments (happy or angry) on emotional faces with varying degrees of ambiguity. For bias training, participants viewed unambiguously angry faces in a task exploiting visual adaptation effects. The results revealed an overall shift from a slightly positive bias in categorizing faces pretraining to a more positive bias after training. Carriers of the deletion variant overall showed a more positive bias than did the noncarriers. Follow-up analyses showed that pretraining bias was a significant predictor of bias change, with those who showed a more negative bias preadaptation changing more in a positive direction. Critically, this effect was observed under control but not under stress conditions. These results suggest that the NE/stress system plays an important role in influencing trait-like biases as well as short-term changes in the tendency to perceive ambiguous stimuli as being more rewarding than threatening.

Acute psychophysiological stress impairs human associative learning.

Addiction is increasingly discussed asa disorder of associative learning processes, with both operant and classical conditioning contributing to the development of maladaptive habits. Stress has long been known to promote drug taking and relapse and has further been shown to shift behavior from goal-directed actions towards more habitual ones. However, it remains to be investigated how acute stress may influence simple associative learning processes that occur before a habit can be established. In the present study, healthy young adults were exposed to either acute stress or a control condition half an hour before performing simple classical and operant conditioning tasks. Psychophysiological measures confirmed successful stress induction. Results of the operant conditioning task revealed reduced instrumental responding under delayed acute stress that resembled behavioral responses to lower levels of reward. The classical conditioning experiment revealed successful conditioning in both experimental groups; however, explicit knowledge of conditioning as indicated by stimulus ratings differentiated the stress and control groups. These findings suggest that operant and classical conditioning are differentially influenced by the delayed effects of acute stress with important implications for the understanding of how new habitual behaviors are initially established.

Dose response of continuous subcutaneous infusion of recombinant glucagon-like peptide-1 in combination with metformin and sulphonylurea over 12 weeks in patients with type 2 diabetes mellitus.

AIMS: Any differences observed between natural glucagon-like peptide-1 (GLP-1) and studies obtained with analogues might call for renewed considerations concerning the use and design of such analogues. Thus, we aimed to evaluate the dose-response relationship of recombinant glucagon-like peptide-1 (7-36) amide (rGLP-1) administered by continuous subcutaneous infusion (CSCI) in subjects with type 2 diabetes. METHODS: We compared the efficacy and safety of three doses of recombinant GLP-1, ranging from 1.25 to 5.0 pmol/kg/min (pkm) and placebo, given by continuous subcutaneous infusion over 3 months in combination with metformin and sulphonylurea (SU), to lower haemoglobin A1c (HbA1c), fasting plasma glucose and weight in 95 type 2 diabetes patients with inadequate glycaemic control. RESULTS: The mean decreases in HbA1c at endpoint (week 12) were significantly greater for all three rGLP-1 dose groups when each was compared with the placebo group, with the greatest decrease occurring in the 5.0 pkm dose group (-1.3%, s.d. ± 0.18, p < 0.001). The mean decreases in fasting plasma glucose from baseline to endpoint were significantly greater for all three rGLP-1 dose groups than for the placebo group, with the greatest decrease occurring in the 5.0 pkm dose group (-26.0 mg/dl, s.d. ± 8.5, p = 0.02). Body weight was significantly reduced by 1.8 kg (s.d. ± 1.3) in the 1.25 pkm dose group only (p = 0.04). CONCLUSIONS: Administration of rGLP-1 by CSCI over a 12-week period in combination with metformin and an SU had a dose dependent effect in lowering HbA1c and fasting plasma glucose. However, administration of rGLP-1 by CSCI may be less effective with respect to lowering of body weight compared with the daily and once weekly analogues.

Dose response of subcutaneous GLP-1 infusion in patients with type 2 diabetes.

AIM: To evaluate the dose-response relationship of the recombinant glucagon-like peptide-1 (7-36) amide (rGLP-1) administered by continuous subcutaneous infusion (CSCI) in subjects with type 2 diabetes, with respect to reductions in fasting, postprandial and 11-h serum glucose profiles. METHODS: In a double-blind, parallel, placebo-controlled trial, 47 patients were randomized to placebo or rGLP-1 (1.25, 2.5, 5.0 or 8.5 pmol/kg/min) by CSCI for 7 days. On day 1 (pretreatment) and on day 8, patients underwent monitoring of fasting, postprandial, and 11-h profiles of glucose and hormones. RESULTS: Fasting serum glucose decreased by 76.2, 53.9, 37.0 and 22.7 mg/dl for the 8.5, 5.0, 2.5 and 1.25 pmol/kg/min rGLP-1 groups, respectively, compared to a decrease of 1.1 mg/dl for placebo (p = 0.0002, 0.005, 0.064 and 0.27, respectively). Mean 11-h serum glucose area under the curve decreased by 36.3, 23.3, 16.9 and 10.0% for 8.5, 5.0, 2.5 and 1.25 pmol/kg/min rGLP-1, respectively, compared to no change for placebo (p = 0.0001, 0.0019, 0.012 and 0.14, respectively). Mean fasting C-peptide increased dose dependently with rGLP-1 (p = 0.0023 for the highest dose) and decreased with placebo. There were no serious safety concerns and no instances of hypoglycaemia. CONCLUSIONS: rGLP-1 produced continuous improvements in glycaemic control across a broad dose range of up to 8.5 pmol/kg/min.

Recombinant glucagon-like peptide-1 (7-36 amide) lowers fasting serum glucose in a broad spectrum of patients with type 2 diabetes.

AIMS: To evaluate the safety and efficacy of various doses of recombinant glucagon-like peptide-1 (7-36) amide (rGLP-1) administered subcutaneously (s. c.) via bolus injection or continuous infusion to lower fasting serum glucose (FSG) levels in subjects with type 2 diabetes treated by diet, hypoglycemic drugs, or insulin injection. METHODS: rGLP-1 was administered s. c. to 40 type 2 diabetics currently treated by diet, sulfonylurea (SU), metformin, or insulin in a double-blind, placebo-controlled, cross-over trial; preexisting treatments were continued during the study. In the bolus injection protocol, 32 subjects (8 from each of the 4 treatment groups) received 0.0, 0.5, 1.0, and 1.5 nmol rGLP-1/kg per injection (two injections, two hours apart, beginning one hour after the evening meal) in a randomized order on separate days. In the continuous s. c. infusion protocol, 40 subjects received rGLP-1 at 0.0, 1.5, 2.5, 3.5, and 4.5 pmol/kg/min for 10-12 hours overnight starting one hour after the evening meal. Fasting bloods were taken the morning after for glucose, insulin, and glucagon measurements. RESULTS: In the diet, SU, and metformin cohorts, bolus rGLP-1 injections produced modest reductions in mean FSG levels, averaging 17.4 mg/dl (7.3-27.5; 95 % CI) at the highest dose (p < 0.001 vs. placebo). Reductions in FSG levels were greater by continuous infusion at up to 30.3 mg/dl (18.8 - 41.8; 95 % CI; p < 0.001 vs. placebo). The greatest reduction in mean FSG occurred in the SU cohort (up to 43.9 mg/dl, 24.7 - 63.1; 95 % CI; p < 0.001). rGLP-1 infusions resulted in significant increases in fasting plasma insulin and decreases in fasting plasma glucagon levels. There were no serious adverse events; GI-related symptoms were dose-related and more commonly associated with injections. CONCLUSIONS: rGLP-1 (7-36) amide dose-dependently lowered FSG in a broad spectrum of type 2 diabetics when added to their existing treatment. Subcutaneous infusion was more effective than injection, and the combination with SU was more effective than with metformin.

Cleavage of disulfide-bridged stalk domains during shedding of angiotensin-converting enzyme occurs at multiple juxtamembrane sites.

Shedding of the ectodomain of angiotensin-converting enzyme (ACE) and numerous other membrane-anchored proteins results from a specific cleavage in the juxtamembrane (JM) stalk, catalyzed by "sheddases" that are commonly activated by phorbol esters and inhibited by peptide hydroxamates such as TAPI. Sheddases require a stalk of minimum length and steric accessibility. However, we recently found that substitution of the ACE stalk with an epidermal growth factor (EGF)-like domain from the low-density lipoprotein receptor (LDL-R) did not abolish shedding; cleavage of the ACE-JMEGF chimera occurred at a Gly-Phe bond in the third disulfide loop of the EGF domain. We have now constructed two additional stalk chimeras, in which the native stalk in ACE was replaced with the EGF domain from factor IX (ACE-JMfIX) and with a cysteine knot motif (ACE-JMmin23). Like the ACE-JMEGF chimera, the ACE-JMfIX and -JMmin23 chimeras were also shed, but mass spectral analysis revealed that the cleavage sites were adjacent to, rather than within, the disulfide-bonded domains. Homology modeling of the LDL-R EGF domain revealed that the third disulfide loop is larger and more flexible than the equivalent loop in the factor IX EGF domain. Similarly, the NMR structure of the Min-23 motif is highly compact. Hence, cleavage within a disulfide-bonded domain appears to require an unhindered loop. Interestingly, unlike wild-type ACE and the ACE-JMEGF and -JMmin23 chimeras, shedding of the ACE-JMfIX chimera was not stimulated by phorbol or inhibited by TAPI, but instead was inhibited by 3,4-dichloroisocoumarin, indicating the activity of an alternative sheddase. In summary, the ACE shedding machinery is highly versatile, but an accessible JM sequence, in the form of a flexible stalk or an exposed loop within or adjacent to a folded domain, appears to be required. Moreover, alternative sheddases are recruited, depending on the nature of the JM sequence.

Roles of the juxtamembrane and extracellular domains of angiotensin-converting enzyme in ectodomain shedding.

Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cleavage by a secretase. To investigate the requirements for ectodomain shedding, we replaced the glycosylphosphatidylinositol addition sequence in membrane dipeptidase (MDP) - a membrane protein that is not shed - with the juxtamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resulting construct, MDP-STM(ACE), was targeted to the cell surface in a glycosylated and enzymically active form, and was shed into the medium. The site of cleavage in MDP-STM(ACE) was identified by MS as the Arg(374)-Ser(375) bond, corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somatic ACE. The release of MDP-STM(ACE) and ACE from the cells was inhibited in an identical manner by batimastat and two other hydroxamic acid-based zinc metallosecretase inhibitors. In contrast, a construct lacking the juxtamembrane stalk, MDP-TM(ACE), although expressed at the cell surface in an enzymically active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACEDeltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cleavable stalk, implying that in contrast with the C-terminal domain, the N-terminal domain lacks a signal required for shedding. These data are discussed in the context of two classes of secretases that differ in their requirements for recognition of substrate proteins.

Recombinant human GHRH(1-44)NH2: clinical utility and therapeutic development program.

Growth hormone (GH) secretagogues are becoming increasingly attractive alternatives to GH or insulin-like growth factor-I (IGF-I) for the treatment of conditions that may benefit from activation of the GH/IGF-I axis. This stems from the realization that (1) GH secretagogues stimulate the pulsatile release of endogenous GH; (2) feedback control of endogenous GH and IGF-I is preserved, guarding against imbalances between GH and IGF-I levels; and (3) GH treatment is associated with adverse effects in the elderly. Of the GH secretagogues, growth hormone-releasing hormone (GHRH) remains the best characterized, in terms of identity of the ligand-receptor pair and its exclusive somatotropic activity at the level of the pituitary. Full-length natural GHRH (1-44) amide can now be produced by recombinant technologly on a commercially viable scale, and is currently being evaluated in early phase clinical trials. The purpose of these studies is to evaluate the efficacy and tolerability of chronic subcutaneous administration of GHRH over a range of doses in elderly subjects. Therapeutic areas that are being investigated in the elderly include congestive heart failure, osteoporosis, and improvements in body composition and function in the frail elderly.

The mycosins of Mycobacterium tuberculosis H37Rv: a family of subtilisin-like serine proteases.

There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of proteases in the pathogenesis of tuberculosis. We identified five M. tuberculosis genes (mycP1-5) that encode a family of serine proteases (mycosins-1 to 5), ranging from 36 to 47% identity. Each protein contains a catalytic triad (Asp, His, Ser) within highly conserved sequences, typical of proteases of the subtilisin family. These genes are also present in M. bovis BCG and other virulent mycobacteria, but only one homologue (mycP3) was detected in M. smegmatis. The mycosins have N-terminal signal sequences and C-terminal transmembrane anchors, and the localisation of the mycosins to the membrane/cell wall was verified by Western blot analysis of heterologously expressed proteins in cellular fractions of M. smegmatis. In M. tuberculosis, all the mycosins were expressed constitutively during growth in broth. Mycosins-2 and 3 were also expressed constitutively in M. bovis BCG, but no expression of mycosin-1 was detected. Mycosin-2 was modified by cleavage in all three mycobacterial species. The multiplicity and constitutive expression of these proteins suggests that they have an important role in the biology of M. tuberculosis.

CR3: a general purpose adhesion-recognition receptor essential for innate immunity.

CR3 (CD11b/CD18), a beta(2) integrin, has a key role in innate antimicrobial defenses, as evidenced by the leukocyte adhesion (CD18) deficiency syndrome in humans and the CD11b knockout mouse. CR3 is a highly versatile pattern-recognition receptor that activates leukocytes via signaling complexes and actin reorganization, mediates phagocytosis, and promotes leukocyte transmigration.

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites.

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg(1203). These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bond, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].

Phorbol ester-induced juxtamembrane cleavage of angiotensin-converting enzyme is not inhibited by a stalk containing intrachain disulfides.

Specialized proteases, referred to as sheddases, secretases, or membrane-protein-solubilizing proteases (MPSPs), solubilize the extracellular domains of diverse membrane proteins by catalyzing a specific cleavage in the juxtamembrane stalk regions of such proteins. A representative MPSP (tumor necrosis factor-alpha convertase) was cloned recently and shown to be a disintegrin metalloprotease that is inhibited by peptide hydroxamates including the compound TAPI. Substrate determinants that specify cleavage by MPSPs remain incompletely characterized, but may include the physicochemical properties of the stalk or unidentified recognition motifs in the stalk or the extracellular domain. We constructed a mutant angiotensin-converting enzyme (ACE) in which the stalk has been replaced with an epidermal growth factor (EGF)-like domain (ACE-JMEGF), to test the hypothesis that MPSP cleavage requires an open, comparatively unfolded or extended stalk. Wild-type ACE is a type I transmembrane (TM) ectoprotein that is efficiently solubilized by a typical MPSP activity. We found that ACE-JMEGF was solubilized inefficiently and accumulated in a cell-associated form on transfected Chinese hamster ovary (CHO) cells; cleavage was stimulated by phorbol ester and inhibited by TAPI, features typical of MPSP activity. Determination of the C-terminus of soluble ACE-JMEGF revealed that, surprisingly, cleavage occurred at a Gly-Phe bond between the fifth and sixth cysteines within the third disulfide loop of the EGF-like domain. Reduction of intact CHO cells with tributylphosphine resulted in the rapid release of ACE-JMEGF (but not wild-type ACE) into the medium, suggesting that a proportion of membrane-bound ACE-JMEGF is cleaved but remains cell-associated via disulfide tethering. The mechanism for the release of ACE-JMEGF in the absence of chemical reduction is unclear. We conclude that the presence of a compact, disulfide-bridged domain does not per se inhibit cleavage by an MPSP activity, but ectodomain release is prevented by disulfide tethering to the TM domain.

Interactions between Mycobacterium tuberculosis and host cells: are mycobacterial sugars the key?

Mycobacterium tuberculosis has evolved successful strategies to invade and persist within macrophages. Intimate pathogen-macrophage contacts dictate receptor choice and probably specify the intracellular fate of these microorganisms. Binding to specific receptors, such as complement receptor type 3, could provide an advantage. These interactions appear to involve surface polysaccharides and glycolipids.

Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent.

The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis. We have identified two substrains of M. tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum. H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside. H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns. Of five M. tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH. Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages. Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M. tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH. Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH. Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv-HH; these differences were eliminated after treatment of H37Rv-CC with glass beads. Thus, outer capsular polysaccharides mediate the binding of H37Rv-CC to CR3, likely to the beta-glucan site. Moreover, there are strain-dependent differences in the thickness or composition of capsular polysaccharides that determine the mode of binding of M. tuberculosis to mammalian cells.

Identification of phosphatidylinositol mannoside as a mycobacterial adhesin mediating both direct and opsonic binding to nonphagocytic mammalian cells.

The molecular basis for the binding of Mycobacterium tuberculosis to nonphagocytic cells, which are readily infected in vitro, and the in vivo significance of this interaction are incompletely understood. Of six cell types tested, we found that only two, Chinese hamster ovary (CHO) fibroblasts and primary porcine aortic endothelial cells, were able to bind M. tuberculosis H37Rv efficiently in vitro. Binding to both CHO and endothelial cells was markedly (three- to fivefold) enhanced by 10 to 20% human or bovine serum, suggesting that the bacteria were coated by a serum opsonin. Preincubation with individual candidate opsonins revealed that recombinant human mannose-binding protein (rMBP), fibronectin, and transferrin were each able to enhance binding threefold. Preincubation of bacteria in serum depleted of mannan-binding lectins or in genetic MBP-deficient serum resulted in enhancements that were only approximately 60 and 58%, respectively, of that produced by preincubation in control serum. In contrast, serum depleted of fibronectin or transferrin retained its opsonizing capacity, suggesting that the latter two are not significant opsonins in whole serum. Binding of M. tuberculosis and Mycobacterium smegmatis to both CHO and endothelial cells in the presence or absence of serum was blocked (60 to 70%) by a monoclonal antibody, MAb 1D1, selected for recognition of intact bacilli. The 1D1 antigen was purified from mycobacterial cell walls and chemically identified as a polar phosphatidylinositol mannoside (PIM). Latex beads coated with purified 1D1 antigen bound to CHO cells, which was enhanced threefold by serum and abolished by periodate treatment, suggesting a requirement for the PIM mannoses in opsonic adhesion. This was likely mediated, at least in part, by serum MBP, as rMBP bound strongly to 1D1 antigen in both thin-layer chromatography overlay and plate binding assays, the latter in a mannan-inhibitable manner. This is the first demonstration that mycobacterial PIMs can function as adhesins for binding to nonphagocytic cells, both directly and after opsonization with serum proteins, including MBP.

Proteolytic release of membrane proteins: studies on a membrane-protein-solubilizing activity in CHO cells.

Diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE) or juxtamembrane (stalk) deletion or chimaera mutants. Determination of the C termini (i.e. the cleavage sites) of released, soluble wild-type and mutant ACE by MALDI-TOF mass spectrometry indicated that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane, but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, kinetic analyses of release rates indicated that a minimum distance from the membrane must be preserved. Interestingly, soluble full-length (anchor-plus) WT-ACE incubated with fractions of, or intact, CHO cells was not cleaved. In all cases, release was stimulated by a media change or by the addition of phorbol ester, with rate enhancements of 5- and 50-fold, respectively, for WT-ACE. The phorbol ester effect was abolished by staurosporine, a protein kinase C (PKC) inhibitor. We propose that the CHO cell MPSP that solubilizes ACE: (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain and (4) is regulated in part by a PKC-dependent mechanism.

Identification of N-linked glycosylation sites in human testis angiotensin-converting enzyme and expression of an active deglycosylated form.

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.

Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells.

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.

Proteolytic release of membrane-bound angiotensin-converting enzyme: role of the juxtamembrane stalk sequence.

Many structurally and functionally diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors, and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE; human testis isozyme) or one of four juxtamembrane (stalk) mutants containing either deletions of 17, 24, and 47 residues (ACE-JM delta 17, -JM delta 24, and -JM delta 47, respectively) or a substitution of 26 stalk residues with a 20-residue sequence from the stalk of the low-density lipoprotein receptor (ACE-JMLDL). The C termini of released, soluble WT-ACE and ACE-JM delta 17 and -JMLDL were determined by MALDI-TOF mass spectrometry analyses of C-terminal peptides generated by CNBr cleavage. Observed masses of 4264 (WT-ACE) and 4269 (ACE-JM delta 17) are in good agreement with an expected mass of 4262 for the C-terminal CNBr peptide ending at Arg-627, indicating cleavage at the Arg-627/Ser-628 bond in both WT-ACE and ACE-JM delta 17, at distances of 24 and 10 residues from the membrane, respectively. Data for ACE-JM delta 24 are also consistent with cleavage at or near Arg-627. For ACE-JMLDL, in which the native cleavage site is absent, observed masses of 4372 and 4542 are in close agreement with expected masses of 4371 and 4542 for peptides ending at Ala-628 and Gly-630, respectively, indicating cleavages at 17 or 15 residues from the membrane. These data indicate that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, cleavage at a distance of 10 residues from the membrane is more favorable, as ACE-JM delta 17 is cleaved 12-fold faster than WT-ACE. In contrast, ACE-JM delta 24 is released 17-fold slower, suggesting that a minimum distance from the membrane must be preserved. This is supported by results with the ACE-JM delta 47 mutant, which is membrane-bound but not cleaved, likely because the entire stalk has been deleted. Finally, soluble full-length (anchor-plus) WT-ACE is not cleaved when incubated with various CHO cell fractions or intact CHO cells. On the basis of these and other data, we propose that the CHO cell MPSP that solubilizes ACE (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain; and (4) may have a weak preference for cleavage at Arg/Lys-X bonds.