Peter Lemansky

Identification of plasma membrane associated mature β‐hexosaminidase A, active towards GM2 ganglioside, in human fibroblasts

Mature β‐hexosaminidase A has been found associated to the external leaflet of plasma membrane of cultured fibroblasts. The plasma membrane association of β‐hexosaminidase A has been directly determined by cell surface biotinylation followed by affinity chromatography purification of the biotinylated proteins, and by immunocytochemistry. The immunological and biochemical characterization of biotinylated β‐hexosaminidase A revealed that the plasma membrane associated enzyme is fully processed, suggesting its lysosomal origin.

Targeting myeloperoxidase to azurophilic granules in HL‐60 cells

Myeloperoxidase (MPO) is a cationic protein and one of the major constituents of azurophilic granules in neutrophils. Here, we examined whether intracellular transport of MPO and serglycin, a chondroitin sulfate (CS)‐bearing proteoglycan, is correlated. First, we examined binding of MPO to CS–Sepharose and measured an ionic interaction, which was disrupted by 200–400 mM NaCl. Next, HL‐60 promyelocytes were activated with a phorbol ester, which induced an almost complete rerouting of serglycin from the granular to the secretory pathway, concomitant with a similar effect on MPO transport and secretion. We then used the membrane‐permeable cross‐linker dithiobis(succininmidylpropionate; DSP) after labeling HL‐60 cells with [35S]methionine and [35S]cysteine for 19 h. Immunoprecipitation of MPO revealed its cross‐linking to high molecular material having the appearance of a proteoglycan in sodium dodecyl sulfate‐polyacrylamide gels. This assumption was confirmed by labeling HL‐60 cells with [35S]sulfate for 10 min followed by DSP cross‐linking and immunoprecipitation. From three granular enzymes immunoprecipitated, only the cationic MPO was cross‐linked to [35S]sulfate‐labeled serglycin in appreciable quantities, whereas cathepsin D or β‐N‐acetylhexosaminidase was not. Thus, intracellular transport of MPO appears to be linked to that of serglycin. Extracts from high buoyant density organelles from human placenta containing MPO activity were subjected to CS‐affinity chromatography. Proteins binding to CS were identified by mass spectrometry as MPO, lactoferrin, cathepsin G, and azurocidin/cationic antimicrobial protein of molecular weight 37 kDa, suggesting that serglycin may be a general transport vehicle for the cationic granular proteins of neutrophils.

The cation-independent mannose 6-phosphate receptor is involved in lysosomal delivery of serglycin

To clarify the sorting mechanism of the lysosomal/granular proteoglycan serglycin, we treated human promonocytic U937 cells with p‐nitrophenyl‐β‐D‐xyloside (PNP‐xyl) and cycloheximide. In the absence of protein synthesis, the carbohydrate moiety of serglycin was synthesized as PNP‐xyl‐chondroitin sulfate (CS), and most of it was delivered to lysosomes and degraded. Further, an augmented lysosomal targeting of serglycin in the presence of tunicamycin suggested that a sorting/lectin receptor with multiple specificity was involved with an increased capacity for serglycin in the absence of N‐glycosylation. Correspondingly, the cation‐independent mannose 6‐phosphate receptor (CI‐MPR) and sortilin were observed to bind to immobilized CS. These receptors were eluted in the presence of 200–400 mM and 100–250 mM NaCl, respectively. After treating the cells with a cross‐linking reagent, a portion of the sulfated proteoglycan was coimmunoprecipitated with the CI‐MPR but not with sortilin. In the presence of phorbol ester, lysosomal targeting of serglycin and to a lesser extent, of cathepsin D was inhibited. We conclude that the CI‐MPR participates in lysosomal and granular targeting of serglycin and basic proteins such as lysozyme associated with the proteoglycan in hematopoietic cells.

DEFECTIVE PROCESSING OF LYSOSOMAL ENZYMES

DEFECTIVE PROCESSING OF LYSOSOMAL ENZYMES Kurt von Figura, Andrej Hasilik, Regina Pohlmann, Peter Lemansky and Thomas Braulke Physiologisch-Chemisches Institut, University of MUnster, D-4400 MUnster, FRG 7. Ginns EI, Brady RO, Pirruccello S, Moore C, Sorrell S, Furbish FS, Murray GJ, Tager J and Barranger JA 1982 Proc Natl Acad Sci U.S.A. 79: 5607 8. Lemansky P, Bishop DF, Desnick RJ, Hasilik A and von Figura K 1986 J BioI Chem submitted 9. Conary J, Beck M, Hasilik A and von Figura K (unpublished)

Transport and Processing of Lysosomal Enzymes

At present more than 50 different lysosomal enzymes are known. They have in common their location in lysosomes and their function to degrade endogenous and exogenous macromolecules mostly by hydrolytic cleavage. With a few exceptions lysosomal enzymes fall into the class of low abundant proteins with a frequency ranging from 0.05 to 0.005% of total protein. Their low frequency severely impairs studies on the in-vitro synthesis of lysosomal enzymes. The best characterized example for in-vitro synthesis of lysosomal enzymes is that of cathepsin D programmed by porcine spleen mRNA (1–3). Cathepsin D is synthesized with an aminoterminal extension of 20 amino acid. This aminoterminal “signal sequence” directs translocation of the nascent cathepsin D precursors across the membrane of the rough endoplasmic reticulum. Translocation involves recognition of the signal sequence when protruding from the ribosomes by a cytosolic ribonucleotide particle (SRP) and transfer of this complex to a receptor protein (SRP-receptor or docking protein) at the cytosolic face of the rough endoplasmic reticulum. After crossing the membrane the signal sequence is cleaved by a proteinase at the luminal side of the rough endoplasmic reticulum. It is therefore not found on cathepsin D, isolated from translation system supplemented with dog pancreas microsomal vesicles. The existence of a similar transient aminoterminal presequence has been postulated for s-glucuronidase and cathepsin D in rat hepatocytes and the α- and s-chain of s-hexosaminidase in human fibroblasts by comparison of the size of the in-vitro synthesized products with that of tunicamycin treated cells (4,5).