The EMBO Journal vol.9 no. 1 1 pp.3507 - 3513, 1990

Mannose 6-phosphate lysosomal enzymes

receptor dependent secretion of

Hanna Huey-Jiun Chao, Abdul Waheed, Regina Pohlmann, Annette Hille and Kurt von Figura Georg-August-Universitiit Gottingen, Biochemie II, Gosslerstrasse 12d, D-3400 Gottingen, FRG Communicated by K.von Figura

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46. Key words: ligand binding/lysosome/mannose 6-phosphate receptor

mediates endocytosis of mannose 6-phosphate containing ligands (Gartung et al., 1985). The inability of cell surface associated MPR 46 to bind ligands and mediate their endocytosis is surprising, since MPR 46 recycles between the cell surface and internal membranes (Stein et al., 1987). The contribution of the MPR 46 to the targeting of newly synthesized lysosomal enzymes becomes apparent only if the MPR 300 is deficient or blocked by antibodies. In such cells secretion of newly synthesized lysosomal enzymes is increased (Gabel et al., 1983; Gartung et al., 1985). Increasing the level of MPR 46 in these cells by transfection of MPR 46 cDNA decreases the secretion (Kornfeld and Mellman, 1989), while the presence of antibodies that block the ligand binding site of MPR 46 increases the secretion (Stein et al., 1987). The lack of cell lines deficient in MPR 46 has hampered the functional analysis of this receptor. In the present study we have analyzed the contribution of MPR 46 to the transport of newly synthesized lysosomal enzymes by transfecting the cDNA for human MPR 46 into hamster and mouse cells, which express basal levels of both MPR 46 and MPR 300. The results of this study provide evidence that part of the newly synthesized lysosomal enzymes that bind to the human MPR 46 become secreted and that the secretion of newly synthesized lysosomal enzymes in non-transfected hamster and mouse cells is mediated-at least in part-by the endogenous MPR 46. -

-ex osaminid a se

BH.(--M D

c

BeK MPR46

u%I c

M

Arylsulfatase A BHK-PM C

M

BHK-MPR46 C

M

._

_NM

Introduction 97 4

Mammalian cells express two mannose 6-phosphate receptors with apparent sizes of 46 000 (MPR 46) and 300 000 (MPR 300). The two receptors mediate the transport of newly synthesized soluble lysosomal proteins to lysosomes. They bind their ligands within the secretory route and transfer them to the endocytic route, where the ligands are released and delivered to lysosomes, while the receptors recycle to the secretory route (von Figura and Hasilik, 1986; Dahms et al., 1989; Kornfeld and Mellman, 1989). With the exception of a few tumor derived cell lines the two receptors are expressed simultaneously in cells and have a similar subcellular distribution (Gabel et al., 1983; Bleekemolen et al., 1988). The two receptors appear to bind the same ligands although with different affinities and pH optima (Hoflack et al., 1987; Tong and Kornfeld, 1989). Two major functional differences between the two receptors have been noted so far. The MPR 300 binds in addition to mannose 6-phosphate the insulin like growth factor II (IGF II) in many but not all species (Morgan et al., 1987) and © Oxford University Press

if ' .,t

4'l

M

6g-t 0

v

In-- I.)

_19f

Om

_

*:

m~~~~~~~

N

46-U

30-

Fig. 1. Secretion of (3-hexosaminidase and arylsulfatase A. BHK-PM and BHK-MPR 46 cells were labeled for 3 h with [35S]methionine and then chased for 6 h. :3-Hexosaminidase and arylsulfatase A were quantitatively immunoprecipitated from extracts of cells (C) and media (M). The position of precursor (p) and proteolytically processed (m) forms of the a- and ,8-chain of ,B-hexosaminidase and of arylsulfatase A are indicated. The assignment of the ca- and 13-chains is made by analogy to the human forms of ,B-hexosaminidase. 14C mol. wt markers

are

shown at the left.

3507

H.H.-J. Chao et al.

Results

6-phosphate (M6P)-containing polypeptides, mostly representing lysosomal enzymes, were retained and eluted with 5 mM mannose 6-phosphate (Figure 2). In BHK-MPR 46 cells the amount of secreted M6P-containing polypeptides 10-fold higher than in BHK-PM or in non-transfected was BHK 21 cells and represented 1% of total [35S]radioactivity incorporated by the cells into trichloroacetic acid insoluble material (Table I). These results clearly indicate that BHKMPR 46 cells have an impaired capacity to retain newly synthesized M6P-containing polypeptides. nose

Increased secretion of newly synthesized lysosomal enzymes in BHK cells expressing human MPR 46 The targeting of newly synthesized ,B-hexosaminidase and arylsulfatase A was compared in BHK cells transfected with vector DNA alone (BHK-PM) or vector DNA carrying the cDNA of human MPR 46 (BHK-MPR 46). In the secretions of BHK-PM cells 11% and 4% of the newly synthesized ,B-hexosaminidase and arylsulfatase A were recovered as high mol. wt precursors (Figure 1). The intracellularly retained enzyme forms were smaller in size than the secreted forms. This indicates that the intracellular forms had undergone proteolytic processing, which is characteristic of lysosomal enzymes that have been transported to the prelysosome/ lysosome compartment (Hasilik and von Figura, 1984). In BHK-MPR 46 cells intracellular retention was severely decreased with 52% and 55% of the newly synthesized 3-hexosaminidase and arylsulfatase A being recovered as precursors in the secretions. To examine whether retention of newly synthesized lysosomal enzymes is generally impaired in BHK-MPR 46 cells, the secretions of metabolically labeled BHK-MPR 46 cells were passed over a MPR 300-Affigel 10 column. ManPM

MPR46

M6

9 7.469

46 '-.

-. (}

-

The secretion of M6P-containing polypeptides is mediated by MPR 46 The mannose 6-phosphate binding site of MPR 46 and MPR 300 can be blocked specifically by polyclonal receptor antisera, which do not cross react with other MPR (Gartung et al., 1985; Stein et al., 1987). Incubation of cells in the presence of a receptor antiserum leads to the blocking of cell surface associated and intracellular forms of the receptor without significantly altering the level of either of the two MPR. Blocking of internal receptors by addition of antiserum to the medium is ascribed to the formation of stable receptor -antibody complexes, when internal receptors cycle

via the cell surface. When BHK-MPR 46 cells were incubated in the presence of 10% MPR 46 antiserum, the secretion of M6P-containing polypeptides was decreased to -60% of that in cells incubated in the presence of 10% preimmune serum (Figure 3, Table I). A similar decrease of secretion was also observed when BHK-MPR 46 cells were incubated in the presence of 27 ,tg/ml affinity purified antibodies against the MPR 46 (not shown). In addition the BHK-MPR 46 cells were incubated in the presence of an antiserum which blocks the mannose 6-phosphate binding site of MPR 300. The presence of 10% MPR 300 antiserum stimulated the secretion in BHK-MPR 46 cells by