Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.32 (bromelain)
 1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human germ cell alkaline phosphatase (GCAP), which shares 98% amino acid sequence identity with the placental AP (PLAP), is expressed by malignant trophoblasts. Protein sequence analysis suggests that the Ser residue at position 92 is the putative active site of GCAP which contains two recognition sequences (Asn122-Thr-Thr124 and Asn249-Arg-Thr251) for asparagine-linked glycosylation. To examine the roles of the Ser residue and glycan moieties on GCAP activity and processing, we altered the GCAP cDNA by site-directed mutagenesis and expressed the GCAP mutants in COS-1 cells. Substitution of Ser-92 with either a Thr (S92T) or an Ala (S92A) residue yielded a GCAP devoid of catalytic activity, suggesting that the Ser codon 92 is the active site of GCAP. Six GCAP mutants that lack one or both glycosylation sites were constructed by substituting either Asn-122 or Asn-249 with an Asp residue or either Thr-124 or Thr-251 with an Ala residue. The mature GCAP migrated as a 65-kDa product, but GCAP mutants lacking one or both glycosylation sites migrated as 62- or 58-kDa polypeptides, respectively, indicating that both sites were glycosylated. All six glycosylated mutants were active enzymatically and, in addition, were equally sensitive to heat, L-leucine, and EDTA inhibition as the parental enzyme. GCAP as well as its two active-site and six glycosylation mutants could be released from the plasma membrane of transfected COS-1 cells by the proteinase bromelain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural and functional analysis of human germ cell alkaline phosphatase by site-specific mutagenesis. 155 93

L-selectin, the peripheral lymph node "homing receptor," is an adhesion protein that mediates lymphocyte binding to lymph node high endothelial venules. Ligands for this protein have been identified only on endothelial cells, and recent murine studies indicate that CD34 on endothelial cells is an L-selectin ligand. To investigate whether CD34 expressed on hematopoietic cells functions as an L-selectin ligand, we used an in vitro binding assay to examine lymphocyte adherence to KG1a, a CD34+ human hematopoietic progenitor cell line. We observed specific L-selectin-mediated adherence of lymphocytes to KG1a: the binding was calcium-dependent, was strictly inhibited by anti-L-selectin antibodies and by carbohydrate ligands of L-selectin, and was abrogated by induction of L-selectin shedding from the lymphocyte membrane by treatment with phorbol esters. However, blocking studies using anti-CD34 antibodies, and experiments using KG1a cells sorted for CD34 expression and COS-7 cells transfected with full-length CD34 cDNA indicate that the ligand on KG1a is not CD34; moreover, RPMI 8402, a CD34+ cell line, does not support lymphocyte adherence in the binding assay. Treatment of KG1a with the enzymes neuraminidase, chymotrypsin, and bromelain abrogated lymphocyte binding to the cells, indicating that the ligand is a glycoprotein. These experiments show that CD34 on hematopoietic cells is not an L-selectin ligand and provide the first evidence of a ligand for L-selectin present on a non-endothelial cell.
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PMID:Detection of an L-selectin ligand on a hematopoietic progenitor cell line. 752 35