Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

Abstract

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70‐fold higher enzyme activities of acid phosphatase than non‐transfected BHK cells. The CT29‐LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans‐Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29‐LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose‐6‐phosphate residues suggested that transport does not involve mannose‐6‐phosphate receptors. Part of the membrane‐associated CT29‐LAP was processed to a soluble form. The mechanism that converts CT29‐LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29‐derived cRNA in the presence of microsomal membranes yielded a CT29‐LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29‐LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29‐LAP is released from the membrane by proteolytic cleavage, which removes a C‐terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.

Structure of the human lysosomal acid phosphatase gene

“…A sequence ladder (lanes 3 -6) was used as size markers. Nucleotidc, nt exon 11 is cleaved by proteolytic processing in the lysosomes, where the transmembrane form is converted to the soluble mature form of LAP [3]. The exact site for processing has not yet been determined, The proteolytic processing involves two cleavages; the first by a cysteine proteinase within the cytoplasmic tail and the second by an aspartyl proteinase within the luminal domain close to the site, where LAP exits from the membrane (S. Gottschalk and A. Waheed, unpublished results).…”

“…The deduced sequence and the expression of the cDNA revealed that LAP is synthesized as a transmembrane protein with a cleavable signal sequence at the N-terminus and a short cytoplasmic tail of 18 amino acids. The membrane-bound precursor is transported independently of mannose 6-phosphate receptors to the lysosomes, where the soluble mature form of LAP is generated by proteolytic processing [3]. LAP shares with other lysosomal membrane glycoproteins the dense glycosylation of the luminal domain, a short cytoplasmic domain, mannose-6-phosphate-receptor-independent targeting and partial localization at the cell surface [4-61. While the primary structure of several lysosomal membrane glycoproteins is known, their functions, expression and gene structures remain to be elucidated.…”

The Unique Endosomal/Lysosomal System of Giardia lamblia

“…For instance, AcPh is soluble in Giardia but exists as a type-I membrane protein containing a YXXØ-type internalization sequence in cells as different as Leishmania and humans (Gottlieb and Dwyer, 1981;Waheed et al, 1988;Shakarian et al, 2002), with transport to the lysosome occurring through several cycles of plasma membrane internalization and recycling. In the lysosome of mammalian cells, the luminal domain of AcPh is processed and released in soluble form (Peters et al, 1990).…”

Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding

“…Oligonucleotide-directed mutagenesis of the codon for [9,10]. In pSVL constructs the inserted DNA was under the control of the SV40 late promoter; in pBHE constructs it was under that of the SV40 early promoter.…”

“…BHK cells were grown for 2-3 weeks in the presence of 5 ,ug of puromycin/ml followed by isolation of stably-transfected clones. Microinjection of cRNA in oocytes P29 cDNA inserted into the EcoRI site of pGEM (Promega Biotec) was transcribed using SP6 polymerase and purified as described [10]. RNA (25 ng) in 50 nl of water was injected into Xenopus laevis oocytes (stage V) as described [11].…”