Ectopic Epididymal Expression of Guinea Pig Intestinal Phospholipase B

Abstract

Guinea pig intestinal phospholipase B is a calciumindependent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A 2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.

Enterophilins, a New Family of Leucine Zipper Proteins Bearing a B30.2 Domain and Associated with Enterocyte Differentiation

“…For enterophilin-1, the asterisk showed the irregularity in repeats due to an alanine insertion at position 114. screened using a polyclonal antibody raised against phospholipase B (18,19). Three identical positive clones (7D1, 8D1, and 9D1) carrying 1.5-kb insertions were isolated and sequenced.…”

“…Phage DNA was denatured (1.5 M NaCl, 0.5 M NaOH) and cross-linked to the membrane by exposure to UV. The 9D1 PstI fragment was used as a probe for hybridization as described previously (19). Positive clones were purified through three runs and recovered as Bluescript plasmids by in vivo excision using ExAssist helper phage and SOLR E. coli (Stratagene).…”

“…Phospholipase B probe is a 386-bp fragment amplified by polymerase chain reaction (19). The glyceraldehyde-3-phosphate dehydrogenase probe (350 bp) was prepared by polymerase chain reaction using human lung cDNA as a template (44).…”

“…The probes were labeled by random primer extension using the Nonaprimer Kit (Appligene Oncor) and [␣-32 P]dCTP from Amersham. RNA electrophoresis and filter hybridization were performed as described previously (19).…”

Purification and Characterization of Phospholipase B fromCandida utilis

“…PLB catalyzes hydrolytic cleavage of both the sn-1 and the sn-2 acylester bonds of glycerophospholipids, and has been found in microorganisms, [1][2][3][4][5] plants, 6) and animal tissues. 7,8) The phopholipid deacylating enzymes characterized so far from yeasts at the molecular level belong to the category of the PLB group. But, the enzymatic properties, such as molecular weight, optimum pH, effects of metal ions, substrate specificity, and biological significance vary widely depending on the species of yeast.…”