Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.

Golgi alpha-mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn(1)M(5)Gn(2) to Gn(1)M(3)Gn(2) during biosynthesis of N-glycans. Previously, we isolated a cDNA encoding a protein homologous to alpha-mannosidase II and designated it alpha-mannosidase IIx. Here, we show by immunocytochemistry that alpha-mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with alpha-mannosidase II, alpha-mannosidase IIx colocalizes with alpha-mannosidase II in COS cells. A protein A fusion of the catalytic domain of alpha-mannosidase IIx hydrolyzes a synthetic substrate, 4-umbelliferyl-alpha-D-mannoside, and this activity is inhibited by swainsonine. [(3)H]glucosamine-labeled Chinese hamster ovary cells overexpressing alpha-mannosidase IIx show a reduction of M(6)Gn(2) and an accumulation of M(4)Gn(2). Structural analysis identified M(4)Gn(2) to be Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. The results suggest that alpha-mannosidase IIx hydrolyzes two peripheral Man alpha 1-->6 and Man alpha 1-->3 residues from [(Man alpha 1-->6)(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, during N-glycan processing.

A peptide mimic of E-selectin ligand inhibits sialyl Lewis X-dependent lung colonization of tumor cells.

Selectins bind to carbohydrate ligands in a calcium-dependent manner and play critical roles in host defense and possibly in tumor metastasis. To isolate peptides that mimic E-selectin ligands, we screened a phage peptide library using E-selectin as a target molecule. This attempt unexpectedly failed, probably because the binding affinity of E-selectin to its ligand is low. We then took an approach that is analogous to the isolation of anti-idiotype antibodies and were able to isolate peptides that bound to anticarbohydrate antibodies recognizing E-selectin ligands. These peptides, enriched for their binding to anti-Lewis A antibody, were found to bind to E-, P- and L-selectins in a calcium-dependent manner. Phage harboring the identified peptide IELLQAR and synthetic peptides having the same sequence inhibited the binding of sialyl Lewis X or sialyl Lewis A oligosaccharides to E-selectin. The adhesion of HL-60 and B16 melanoma cells expressing sialyl Lewis X to E-selectin was also inhibited by the phage-displaying IELLQAR peptide. Moreover, i.v. injected IELLQAR peptide inhibited the lung colonization of mouse B16 melanoma and human lung tumor cells expressing sialyl Lewis X. These results demonstrate that it is possible to isolate peptides mimicking carbohydrate ligands by screening the peptides for binding to anticarbohydrate antibodies and then using them to inhibit carbohydrate-dependent experimental tumor metastasis.

Suppression of noxious stimulus-evoked expression of Fos protein-like immunoreactivity in rat spinal cord by a selective cannabinoid agonist.

In rats, cannabinoids inhibit behavioral responses to noxious stimulation with a potency and efficacy similar to that of morphine. However, because cannabinoids depress motor function, it has not been possible to state beyond any doubt that these effects were related to a dampening of noxious sensory input. Therefore, c-fos immunocytochemistry was used to explore the possibility that cannabinoids reduce behavioral responses to noxious stimuli by decreasing spinal processing of nociceptive inputs. Rats received systemic injections of the potent and selective cannabinoid agonist WIN 55,212-2, the receptor-inactive enantiomer WIN 55,212-3 or vehicle prior to observations in a model of tonic pain, the formalin test. As demonstrated previously, plantar injections of formalin led to lifting and licking of the injected paw, with two peaks of activity occurring at 5 and 30 min after injection. The cannabinoid agonist suppressed these pain responses and produced a reduction in mobility. Immunocytochemical processing of sections with an antibody to the Fos protein revealed that the cannabinoid markedly suppressed pain-evoked c-fos expression in the superficial and neck regions of the spinal dorsal horn, but not in the nucleus proprius. Decreased expression of c-fos also occurred in the ventral horn. The specificity of this effect and its probable mediation by cannabinoid receptors are suggested by three findings: (i) the suppression by the drug of both behavioral and immunocytochemical responses to pain was dose-dependent; (ii) neither the behavioral nor the immunocytochemical response to the noxious stimulus was significantly affected by the receptor-inactive enantiomer of the agonist; (iii) animals rendered tolerant to cannabinoids by repeated injections of the agonist showed reduced responses to the drug. These findings suggest that cannabinoids inhibit the spinal processing of nociceptive stimuli and support the notion that endogenous cannabinoids may act naturally to modify pain trnasmission within the central nervous system.