isolation and characterization of human lysosomal membrane ... · matteson, j., and carlsson, s. r....

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 35, Issue of December 15, pp. 18911-18919, 1988 Printed in U.S.A.

Isolation and Characterization of Human Lysosomal Membrane Glycoproteins, h-lamp- 1 and h-lamp-2 MAJOR SIALOGLYCOPROTEINS CARRYING POLYLACTOSAMINOGLYCAN*

(Received for publication, February 18, 1988, and in revised form, June 7, 1988)

Sven R. Carlsson$$, Jurgen Rothll, Friedrich Pillerz, and Minoru FukudaSII From the $ L a Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037, the Department of Physiological Chemistry, §University of Umei, S-90187 Umei, Sweden, and the (Interdepartmental Electron Microscopy, Bwcenter, University of Basel, CH-4056, Basel, Switzerland

Two major lysosomal membrane glycoproteins with apparent M, - 120,000 were purified from chronic myelogenous leukemia cells. These glycoproteins are major sialoglycoproteins containing polylactosaminoglycan and represent approximately 0.1-0.2% of total cell proteins. A monoclonal antibody specific to one of the glycoproteins and polyclonal antibodies specific to the other glycoprotein were obtained. Immunoelectron microscopic examination of HeLa cells revealed that these two glycoproteins mainly reside in lysosomes and multivesicular bodies. Immunoprecipitation experiments showed that a number of different cell lines express these glycoproteins. However, the apparent molecular weights differed between cell lines; this probably represents differences in the amount of polylactosaminoglycan expressed by each cell line. As shown in the following paper (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S . R. (1988) J. Biol. Chern. 263, 18920-18928) one of the glycoproteins is very homologous to that of a mouse counterpart, m-lamp- 1. The human form of this glycoprotein is therefore named human lamp-1 (h-lamp-1), while the other glycoprotein, to which the monoclonal antibody was made, is called human lamp-2 (h-lamp-2).

Pulse-chase labeling experiments detected that h- lamp-1 and h-lamp-2 are produced first as precursor forms of 87.5 and 84 kDa, and treatment with endo-8- N-acetylglucosaminidase H (endo-H) or endo-/3-N-acetylglucosaminidase F (endo-F) reduced their molecular masses to 39.5 and 41.5 kDa, respectively. It was estimated that h-lamp-1 has 18 N-linked saccharides and h-lamp-2 16, based on the results of partial digestions with endo-F. These results indicate that the two lysosomal membrane glycoproteins are extensively modified by N-glycans, and some of these were found to have polylactosaminyl repeats and sialic acid. Hu- man lamp-1 and lamp-2, therefore, serve as good models for understanding polylactosaminoglycan for- mation and the biosynthesis and processing of polylactosaminoglycan-containing glycoprotein.

* This work was supported by Grant CA28896 from the National Cancer Institute (to M. F.), Grant B87-03X-07886-01 from the Swed- ish Medical Research Council (to S. R. C.), and Grant 3.396-086 from the Swiss National Science Foundation and the Kanton Basel-Studt (to J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I( To whom correspondence should be addressed La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.

Polylactosaminoglycans are heterogenous saccharides often having high molecular weights. They are distinguished from complex-type N-linked saccharides by having long side chains of Gal(31+4GlcNAc(31--*3 repeats, which are susceptible to endo-(3-galactosidase (for review see Ref. 1). Polylactosami- noglycans carry various antigenic structures such as AB0 blood group antigens (2, 3), developmental antigens such as mouse F9 antigens (4) and human fetal (i) erythrocyte antigen ( 5 ) , and tumor-associated antigens such as sialyl Le” (6). More recently, it has been shown that the lack of polylactosaminoglycan on the human erythrocyte anion transporter (Band 3) causes the glycoprotein to aggregate, resulting in abnormal membrane structures in a congenital dyserythropoietic ane- mia-type I1 (7, 8).

In human erythrocytes, polylactosaminoglycans are attached to Band 3 and Band 4.5 (which includes glucose transporter) (2, 3, 5 ) , but not to glycophorins (9, 10). On the other hand, little is known about the protein carriers for polylactosaminoglycan in nucleated cells. Since only a limited number of glycoproteins contain polylactosaminoglycan, as shown on PA-1 human teratocarcinoma cells ( l l) , i t is likely that the nature of a protein determines whether it is modified by polylactosaminoglycan.

We have shown previously that glycoproteins with M, - 120,000 are major carriers for polylactosaminoglycan in granulocytic cells (12). In order to characterize the molecules which carry polylactosaminoglycan in nucleated cells, we isolated those glycoproteins. The data presented in this report indicate that these glycoproteins are lysosomal membrane glycoproteins, a group of proteins extensively glycosylated by N-linked saccharides.

EXPERIMENTAL PROCEDURES’

RESULTS

Purification of Lysosomal Membrane Glycoproteins and Pro- duction of Specific Antibodies-We have previously shown that sialoglycoproteins with M, - 120,000 in granulocytic cells contain significant amounts of polylactosaminoglycan (12). In order to isolate the glycoprotein(s), total cell lysates were applied to a column of wheat germ agglutinin-agarose, and the bound glycoproteins were eluted with 100 mM N-acetyl- glucosamine. The eluted glycoproteins were subjected to pre-

Portions of this paper (including “Experimental Procedures,” Tables I and 11, and Figs. 2, 4, 7, and 12) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

18911

18912 Polylactosaminoglycan-containing Lysosomal Membrane Glycoproteins

A 0 C D

kDa

200-

116- m v 92.5-

66-

45-

30-

20-

FIG. 1. Lysosomal membrane glycoproteins (lanes A and B ) and leukosialin (lanes C and D ) before and after endo-@- galactosidase treatment. The glycoprotein fraction containing human lamp-I and lamp-2 and leukosialin were labeled with '*'I. The labeled glycoproteins were incubated with (lunes B and D) and without (lanes A and C) endo-B-galactosidase and subjected to SDS- polyacrylamide gel electrophoresis (7.5-12.5% aceylamide gradient). Gels were subjected to autoradiography.

parative SDS2 gel electrophoresis. The material migrating between M, -100,000 and -130,000 was eluted from gels and subjected to affinity chromatography with anti-leukosialin antibodies coupled to Sepharose. The glycoprotein fraction, which was not bound to the column, appeared to be homoge- nous when analyzed by SDS gel electrophoresis (Fig. 1, lane A ) . This purified glycoprotein fraction was initially used for immunization of a rabbit. The glycoproteins were isolated from the total lysate, but further analysis showed them to be lysosome-associated membrane proteins (lamp) with M, - 120,000. As shown below, this purified glycoprotein fraction contained two lysosomal membrane glycoproteins with similar molecular weights, and the antibodies produced were found to be specific to two lysosomal membrane glycoproteins, h- lamp-1 and h-lamp-2 (see also Ref. 29).

Lysosomal Membrane Glycoproteins Contain Saccharides Susceptible to Endo-P-galactosidase-The lysosomal membrane glycoprotein fraction was susceptible to endo-p-galactosidase treatment (Fig. 1, lanes A and B), indicating that the glycoprotein contains polylactosaminoglycan (1, 8). In contrast, leukosialin isolated from the same CML cells was barely affected by endo-&galactosidase treatment (Fig. 1, lanes C and D). The mobility of the lysosomal membrane glycoproteins in SDS-polyacrylamide gels was not significantly altered after sialidase treatment (Fig. 2, lanes C and G), while leukosialin was found as a more slowly migrating band after sialidase treatment (Fig. 2, lanes D and H). This increase of apparent molecular weight is characteristic of glycoproteins containing a high amount of 0-linked saccharides, as shown previously (14). These results suggest that lysosomal membrane glycoproteins and leukosialin differ significantly in glycosylation. Neither the lysosomal membrane glycoproteins nor leukosialin were affected greatly in mobility by reduction of disulfide bonds (Fig. 2, lanes E-H), indicating monomeric structures of the glycoproteins.

Detection of Lysosomal Membrane Glycoproteins in Various Human Cell Lines-A number of human cell lines were metabolically labeled with [3H]glucosamine and subjected to immunoprecipitation with the lamp-specific antibodies. Immu- noprecipitates were then analyzed by SDS gel electrophoresis

The abbreviations used are: SDS, sodium dodecyl sulfate; CML, chronic myelogenous leukemia; h-lamp-1 and h-lamp-2, human lysosome-associated membrane protein-1 and protein-2; PMSF, phen- ylmethanesulfonyl fluoride; PBS, phosphate-buffered saline; endo-H, endo-0-N-acetylglucosaminidase H; endo-F, endo-0-N-acetylglucosaminidase F.

before and after endo-&galactosidase treatment. As shown in Fig. 3, all of the cell lines tested contain the lysosomal membrane glycoproteins and two additional points were noted 1) the molecular weights of the glycoproteins differ significantly among different cell lines, and 2) the susceptibility to endo-@-galactosidase differs among cell lines tested. The latter characteristic appears to be directly correlated to the level of polylactosamine addition expressed by the different cell lines (see below). The glycoproteins were detected in all cells tested except erythrocytes.

Detection of Precursors of Lysosomal Membrane Glycopro- teins in HL-60 Cells-In order to obtain information on the polypeptide and carbohydrate moieties of the lysosomal membrane glycoproteins, pulse-chase experiments were carried out. Fig. 4A shows that a precursor form of -85 kDa was detected immediately after the pulse labeling, and this precursor form was gradually converted to the mature form with a smeared, heterogenously migrating band of M, - 125,000. The conversion of the precursor to mature forms was found to have tM - 45 min. An apparent heterogeneity in the time required for processing of the precursor form to the mature form was noticed, and a small amount of the precursor form was still detected after 120 min of chase. I t was also noted that the precursor form appeared to consist of two closely spaced bands (Fig. 4A). In order to elucidate the manner in which the precursor and mature forms are glycosylated, the same pulse-labeled glycoproteins were digested with endo-H to remove N-linked carbohydrates. Fig. 4B shows that the precursor form was converted to two bands with apparent molecular weights of -41,500 and -39,500. As shown by its susceptibility to endo-H (compare a t 120 min with or without endo-H), the mature form obtained after 120 min chase still contained a small number of high-mannose saccharides.

In order to estimate how many N-linked saccharides are present in the glycoproteins, the precursor form was digested with endo-F for increasing periods of time. From the results shown in Fig. 5,19 bands were counted from the upper bands of the precursor to the lowest band after digestion, whereas 17 bands were counted from the lower band of the precursor to the second lowest band after digestion. These results, obtained on both HL-60 and K562 cells, suggest that the glycoproteins contain 16 to 18 N-linked saccharides, as fur-

HL-60 K662 HCT-16 PA-l HEp-02 IMR-90

' 1 2 3 ' 1 2 3 ' 1 2 3 ' 1 2 3 ' 1 2 3 ' 1 2 3 ' kDa

200-

116- 92.5-

66-

45-

FIG. 3. Lysosomal membrane glycoproteins from different cell lines after treatment with (lanes 3) or without (lanes 2) endo-@-galactosidase. The glycoproteins were immunoprecipitated by anti-(h-lamp-1 + h-lamp-2) serum from the indicated cell lines which were metabolically labeled with [3H]glucosamine. The immu- noprecipitates were either untreated (lunes 2) or treated (lanes 3) with endo-0-galactosidase. The samples were analyzed by SDS-polyacrylamide gel electrophoresis (9% acrylamide gel) followed by fluo- rography. As controls, cell lysates were immunoprecipitated with normal rabbit serum (lanes I).

Polylactosaminoglycan-containing Lysosomal Membrane Glycoproteins 18913 HL-60 K562

1 2 3 4 5 1 2 3 4 5

kDa kDa ~ "

92.5-

66- 3

45- -39.5

30-

FIG. 5. Partial digestions with endo-F of lysosomal membrane glycoprotein precursor forms from HL-60 and K562 cells. Cells were labeled with [35S]methionine for 10 min and chased with unlabeled methionine for 10 min. Lysosomal membrane glycoprotein precursors were immunoprecipitated with anti-(h-lamp-l and h-lamp-2), and were untreated (lane I ), or treated with endo-F for 5 min (lane 2 ) , 20 min (lane 3 ) , 45 min (lane 4 ) and 24 h (lane 5) . Similar results were obtained when treated with endo-H. Open arrow shows unrelated coprecipitated radioactive material.

A 6 C

1 2 3 1 2 3 1 2 3 kDa

200.

92.5- 116- - - 66- - 45-

- 0 - FIG. 6. Immunoprecipitation of human lamp-1 and lamp-2

and their precursors. HL-60 cells were labeled with [35S]methionine for 10 min, and the label was chased with unlabeled methionine for 10 min (B and C) or for 120 min (A ) . Cell lysates were immunoprecipitated with anti-(h-lamp-2) antibody (lanes 3 ) followed by anti- (h-lamp-1) antibodies (lanes 2) . H-lamp-1 and h-lamp-2 were coim- munoprecipitated with anti-(h-lamp-1 + h-lamp-2) antibodies (lanes 1 ). The glycoproteins were subjected to SDS gel electrophoresis after treatment with (C) or without (A and E ) endo-H digestion.

ther discussed below. The results also suggest that there is no difference in the number of N-glycans attached to lamps between HL-60 and K562 cells.

Isolation of Two Lysosomal Membrane Glycoproteins from the Purified Glycoprotein Fraction-The results shown in Figs. 4 and 5 suggested that the purified glycoprotein fraction actually consists of two different lysosomal membrane glycoproteins. In fact, the monoclonal antibody, obtained by im- munizing with the purified glycoprotein fraction, was found to react with only one of the precursors before endo-H digestion in an immunoprecipitation assay (Fig. 6B, lane 3). We have designated the glycoprotein which reacts with the monoclonal antibody h-lamp-2, and the one which does not react, h-lamp-1. This nomination is based on the findings that human lamp-1 has an amino acid sequence homologous to lamp-1 isolated from mouse (see the following paper, Ref. 30). Before endo-H digestion, the precursor of h-lamp-2 migrated faster (lower MI) than that of h-lamp-1 (Fig. 6B). After endo-

kDa

-92.5

-66

-45 "

-30

H digestion, however, h-lamp-2 migrated slower (greater Mr) than h-lamp-1 (Fig. 6C). Based on the difference in molecular weights before and after endo-H digestion, it was deduced that human lamp-1 and lamp-2 contain 18 and 16 N-linked oligosaccharide chains, respectively.

Isolation of h-lamp-1 and h-lamp-2 by Affinity Chromutog- raphy Employing a Monoclonal Antibody-The anti-(h-lamp- 2) monoclonal antibody was coupled to Sepharose, and the purified glycoprotein fraction, which contained human lamp- 1 and lamp-2, was applied to the column. The glycoprotein, h-lamp-1, which was not bound, was pooled and purified further by Sephacryl S-300 gel filtration. The glycoprotein eluted from the column, h-lamp-2, was also subsequently purified by gel filtration. Each purified glycoprotein migrated as a single broad bandwhen examined by SDS gel electrophoresis. The glycoproteins were detected more easily by periodate-Schiff reaction than by Coomassie Blue staining (Fig. 7). Furthermore, purified lamp-2 showed a higher apparent molecular weight than lamp-1. Each of the purified glycoproteins was subjected to Edman degradation for determin- ing its NHp-terminal sequence, and both preparations showed a single amino acid in each step of Edman degradation, confirming that each glycoprotein was highly purified (Table I).

Amino Acid and Carbohydrate Composition of Human lump- 1 and lamp-2-Table I summarizes the characteristics of human lamp-1 and lamp-2. Both glycoproteins contain large amounts of carbohydrate which contribute to about 60% of each protein. The carbohydrate composition indicates that the glycoproteins contain mainly N-linked saccharides with possibly a small amount of 0-linked saccharides, since N- acetylgalactosamine was detected. Both also contain a significant amount of sialic acid. Both lamp-1 and lamp-2 contain cysteine or cystine, while lamp-1 contains more methionine but less aspartic acid (+asparagine) than lamp-1 (Table 11).

We believe that the glycoprotein fraction not bound by anti-leukosialin antibody (the material shown in Fig. lA) consists of only lamp-1 and lamp-2 for the following reasons. First, the amino acid compositions of the starting material is clearly in ag-reement with the average of lamp-1 and lamp-2 (Table 11). Second, the amino acid sequences obtained in the starting material gave 2 residues in each step, the same residues which were found in purified lamp-1 and lamp-2.

Immunolocalization of Lysosomal Membrane Glycopro- teins-Application of the two rabbit antisera, anti-(lamp-1 + lamp-2) and anti-(lamp-1) to ultrathin sections from HeLa or MCF7 cells yielded the same pattern of staining. However, the anti-(lamp-1 + lamp-2) serum gave consistently more intense labeling over all positive structures. Specific immunolabeling was observed over lysosomes (as evidenced by cytochemically detectable acid phosphatase activity), greatly varying in size and shape, and found either adjacent to the trans side of the Golgi apparatus or in the remaining cyto- plasm (Figs. 8, A and C, and 9, A and B). Gold particles were preferentially located at the luminal side of the lysosomal membrane as well as over amorphous material present in the lysosome lumen.

In parallel to these experiments, the Datura stramonium lectin was employed in a cytochemical affinity technique for the detection of N-acetyllactosaminyl residues in lysosomes. It has been shown that D. stramonium lectin binds to N- acetyllactosaminyl residues and poly-N-acetyllactosamine (25, 26). As shown in Fig. 8, B and D, such residues were detectable at the luminal side of the limiting membrane of variously sized and shaped lysosomes, as well as over the amorphous content material. In similar types of lysosomes


t

FIG. 8. Comparison of the distribution of human lamp- 1 and lamp-2 immunoreactivity and N-acetyllactosaminyl residues in lysosomes of HeLa cells. In A , a group of lysosomes with immunolabel for lamp-1 and lamp-2 at the luminal side of the limiting membrane ( I ) or both the limiting membrane and content of the lumen (2) is shown. Structurally corresponding lysosomes (marked I and 2), but stained for N-acetyllactosaminyl residues with the D. stramonium lectin, are presented in B. Note the similar distribution of gold particles which is also evident from other two micrographs shown in C, lamp-1 and lamp-2 immunoreactivity, and D, N-acetyllactosaminyl residues. Anti-(h-lamp-1 + h-lamp-2) serum was used in A and C. Magnification: A , X24,600; B, X28,200; C, X33,600; D, X27,600.

the pattern of immunolabeling for lysosomal membrane glycoproteins corresponded strikingly to the distribution of N- acetyllactosaminyl residues (compare Fig. 8, A with B, and C with D).

In addition to lysosomes, other subcellular compartments showed positive labeling of gold particles. Multivesicular bodies exhibited specific labeling along their limiting membrane as well as at the level of the internal vesicles (Fig. 9C). Structures resembling peripheral endosomes were free of gold particles, although irregularly shaped vesicular structures closely located to them were labeled (Fig. 9, D and E ) . Plasma membrane labeling was only observed with anti-(lamp-1 + lamp-2) serum (Fig. 9B). This antiserum gave also weak immunolabeling over the trans side of the Golgi apparatus (Figs. 8C and 9F). No gold particles were detectable over the nucleus, mitochondria, or endoplasmic reticulum. When the antiserum was replaced by the preimmune serum, no gold particles were observed (not shown) confirming the specificity of the above described immunolabel.

H-lamp-:! Derived from Different Cell Lines Also Shows Different Molecular Weights-As shown above, the apparent molecular weight of mature h-lamp-2 is larger than h-lamp- 1. The results shown in Fig. 3 could be, therefore, due to differences in the relative proportion of human lamp-1 and

FIG. 9. Immunolocalization of immunoreactivity for human lamp-1 and lamp-2 on Lowicryl K4M thin sections of HeLa cells with the protein A-gold technique. Gold particles are present at the limiting membrane and content of various lysosomal bodies ( A and B ) and multivescular bodies ( C ) . Vesicular structures with an electron-dense content (arrowheads in D and E ) are labeled, whereas other, lucent vesicles are free of gold particles (arrows in D and E ) . Plasma membrane labeling (arrow), preferentially associated with microvilli (arrowheads), and a positive lysosome are shown in B. The Golgi apparatus exhibits weak immunolabeling at the trans side and near small lysosomal bodies ( F ) . Open arrow denotes plasma membrane. Anti-(h-lamp-1 + h-lamp-2) serum was used in A, B, and F, whereas anti-(h-lamp-1) serum was used in C, D, and E. Magnifi- cations: A , X21,300; B, X29,100; C, X29,lOO; D, X38,20O; E, X57,700; F, X44,200.

lamp-2 in different cells. In order to test this possibility, h- lamp-2 produced in various cells was immunoprecipitated by the monoclonal antibody. As shown in Fig. 10, h-lamp-2 molecules from various cell lines exhibit significantly different molecular weights. These results indicate that both lamp-1 and lamp-2 give different molecular weights depending on cell types.

Characterization of Carbohydrate Moiety of Human lamp-1 and lamp-2-In order to preliminarily characterize the carbohydrate moieties of human lamp-1 and lamp-2, glycoproteins were obtained by sequential immunoprecipitation of [3H]glucosamine-labeled K562 and HL-60 cells. After Pronase digestion and Sephadex G-50 gel filtration, glycopeptides obtained from lamp-1 of K562 cells showed essentially two peaks: the glycopeptides which eluted at the void volume (fractions 28-40) and those eluted at fractions 50-70 (Fig. 1I.A). The two glycopeptide fractions were separately subjected to mild alkaline degradation. The glycopeptides eluted at the void volume were converted to saccharides with low molecular weights after this treatment (Fig. 12A), indicating that these saccharides are 0-linked oligosaccharides. When the 0-linked oligosaccharides were applied to a Bio-Gel P-4 column, they eluted at the positions consistent with NeuNAca2~3Gal/3l+3(NeuNAca2-~6)GalNAcOH and NeuNaca2+6(Galpl+3)GalNAcOH (data not shown). In contrast, the glycopeptides which eluted in fractions 50-70 of

Polylactosamimglycan-containing Lysosomal Membrane Glycoproteins 18915

kDa 200-

116- 92.5-

66-

45-

FIG. 10. Immunoprecipitation of human lamp-2 from various cell lines. Human lamp-2 was immunoprecipitated by CD3 antibody from indicated cell lines which were metabolically labeled with [3H]glucosamine. Immunoprecipitation was isolated by using biotin-avidin interaction. The samples were analyzed by SDS-polyacrylamide gel electrophoresis (9% acrylamide gel) followed by fluo- rography. For all cell lines except U266 and Namalva, a volume of lysate containing 1 X lo6 cpm was immunoprecipitated. The lysates of U266 and Namalva contained one-third of the radioactivity.

2 -

30 40 50 60 70 80 90 30 40 50 60 70 80 90

Fraction Number

FIG. 11. Gel filtration of glycopeptides obtained from human lamp-1 and lamp-2 of K562 and HL-60 cells. Pronase digests from h-lamp-1 (A and C ) and h-lamp-2 ( B and D) were subjected to Sephadex G-50 gel filtration. The glycopeptides, indicated by horizontal arrows, were subjected to endo-8-galactosidase digestion ( E and F) or endo-8-N-acetylglucosaminidase H (see Fig. 12) and applied to the same column. A, K562 lamp-1; B, K562 lamp- 2; C,E, HL-60 lamp-1; D,F, HL-60 lamp-2. The elution positions of Band 3 polylactosaminoglycan ( M , -11,000), fetuin N-linked glycopeptides ( M , -2,900), and IgG glycopeptides (MI -1,700) are indicated by vertical arrows (Band 3, Fet, IgG). 3 denotes the elution positions of Gal@1+4GlcNAc~1+3Gal.

the Sephadex G-50 column were not changed after the same treatment (data not shown), and the elution positions of these glycopeptides are essentially the same as those eluted from fetuin. Similar results were obtained on the glycopeptides from lamp-2 of K562 cells (Fig. 11B).

The glycopeptides obtained from lamp-2 of HL-60 cells, on the other hand, provided a different elution profile on Seph- adex G-50 column chromatography. Three peaks were observed, and the second peak, which represents sialylyated N- glycans (indicated by a) , eluted earlier than those obtained

from K562 cells (Fig. 1lD) and these glycopeptides were susceptible to endo-&galactosidase treatment (Fig. 11F). In contrast, lamp-1 and lamp-2 glycopeptides of K562 cells showed minimal susceptibility to endo-@-galactosidase treatment (data not shown). When the 0-linked saccharides from HL-60 lamp-2 were subjected to Bio-Gel P-4 gel filtration, they eluted at the positions corresponding to NeuNAca2- 3Ga1~1~4GlcNAc~1~6(NeuNAca2+3Gal~l+3)Gal- NAcOH and Gal/31+4GlcNAc/31+6 (NeuNAccu2+3GalSl+ 3)GalNAcOH (Fig. 12B). Finally, the glycopeptides b eluted later than IgG glycopeptides, and they were susceptible to endo-H to yield high-mannose oligosaccharides which eluted between fractions 66 and 84 (Fig. 12C). These results indicate glycopeptides b are high-mannose saccharides.

The glycopeptides obtained from HL-60 lamp-1 showed an intermediate elution profile between K562 lamp-2 and HL-60 lamp-2 glycopeptides. The N-glycans of HL-60 lamp-1 ( a of Fig. 1lC) are not as large as those of HL-60 lamp-2 and were only partially digested by endo-&galactosidase (Fig. 11E). These results suggested that both lamp-1 and lamp-2 of HL- 60 cells contain much more polylactosaminoglycan than those of K562 cells, and lamp-2 contains more polylactosaminoglycan than lamp-1 in HL-60 cells. The latter data explain why mature lamp-2 is larger than mature lamp-1, even though the precursor of lamp-2 is smaller than that of lamp-1. These results also indicate that human lamp-1 and lamp-2 contain 0-linked saccharides and high-mannose saccharides in addition to sialylated N-linked saccharides, some of which are polylactosaminoglycans.

DISCUSSION

The present report describes the isolation, characterization, and distribution of two human lysosomal membrane glycoproteins, h-lamp-l and h-lamp-2. These two glycoproteins have polypeptide portions of about 40-kDa and are heavily glycosylated by N-glycans; h-lamp-1 and h-lamp-2 contain 18 and 16 N-glycans, respectively. Most strikingly, some of the N-glycans are polylactosaminoglycans. It is interesting to note that the pattern of distribution for lamps and D. stramonium lectin binding sites is strikingly similar (Fig. 8). In both cases, a spot-like labeling is often seen along the luminal side of the lysosomal membrane. These results are consistent with the findings that D. stramonium lectin preferentially binds to poly-N-acetyllactosaminyl structures (25). Thus, our results obtained by cytochemistry and characterization of saccharides attached to h-lamp-1 and h-lamp-2 agree well and point toward the fact that human lamp-1 and lamp-2 are major sialoglycoproteins carrying polylactosaminoglycan. Similar glycoproteins have previously been detected by monoclonal antibodies in mouse (31, 32), rat (33, 34), and chicken (35, 36) cells. However, the previous studies did not characterize lysosomal membrane glycoproteins with M, - 120,000 as carriers for polylactosaminoglycan.

It is noteworthy that human lamp-1 and lamp-2 exhibit significantly different molecular weights depending on cell types (Figs. 3 and 10). For example, lamps from HL-60 cells showed much higher molecular weights than those from K562 cells. As shown in Fig. 5, however, the lamps contain the same number of N-glycans regardless of cell types. The results shown in Figs. 11 and 12 demonstrate that lamps from HL- 60 cells contain a significant amount of polylactosaminoglycan, whereas lamps from K562 cells essentially lack polylactosaminoglycan. These combined results indicate that the variation in molecular weight is most likely due to differential processing, particularly in the attachment of polylactosaminoglycan. The present results on the content of polylactos-


aminoglycan in lamp-1 and lamp-2 are also consistent with those reported previously on total cellular glycopeptides. It has been shown that K562 cells have minimal amounts of polylactosaminoglycan (37), whereas granulocytic cells (38), HL-60 cells (39), and PA-1 cells (11) express significant amounts of polylactosaminoglycan. Very recently, we have succeeded in isolating cDNAs for human lamp-1 (29, 30) and lamp-2 (30) and the sequences of those cDNAs are consistent with the conclusion that human lamp-1 and lamp-2 contain, respectively, 18 and 16 N-glycosylation sites. Furthermore, the attachment of polylactosamine at some of the glycosylation sites, make the carbohydrate moieties relatively bulky. Since the major portions of the molecules presumably reside in the luminal side of lysosomes, this large carbohydrate moiety probably serves to protect lysosomal membrane glycoproteins from degradation by lysosomal proteases. Experi- ments have also shown that h-lamp-1 and h-lamp-2 indeed are unusually resistant to a variety of different proteases (data not shown).

The isolated glycoproteins contain a significant amount of polylactosaminoglycan and sialic acid, suggesting that the glycoprotein traverses the trans-Golgi cisternae (40). It is unlikely that the carbohydrate moiety is serving as a marker for targeting the molecules to lysosome, since tunicamycin treatment does not inhibit the transport of the glycoproteins to lysosomes (34). In this context, it is interesting to compare the present results with those obtained on mannose-6-phosphate receptors. As shown previously (41, 42), the receptor- lysosomal enzyme complex exits from the Golgi apparatus, most likely from the trans-Golgi. Lysosomal enzymes have been shown to contain mannose 6-phosphate modification of carbohydrates, and a specific receptor for this structure serves to route the enzymes toward the lysosome (43, 44). The receptor-enzyme complex is transported to a prelysosomal compartment where the enzymes are dissociated from the receptors (41, 42). The receptors then recycle to the Golgi apparatus and repeat the process, and thus, the mannose 6- phosphate receptors do not reach lysosomes. On the other hand, lysosomal membrane glycoproteins are mainly present in lysosomes and much less are found in other compartments. These results suggest that a molecular signal for targeting of lysosomal membrane glycoproteins to lysosomes is different from that for mannose 6-phosphate receptor routing. In fact, no similarity exists in amino acid sequences between lysosomal membrane glycoproteins (see the following paper (30)) and mannose 6-phosphate receptors (45-48). Lysosomal membrane glycoproteins can also be detected in plasma membranes, multivesicular bodies, and possibly the trans-Golgi (Figs. 8 and 9; see also Refs. 32, 36, and 49). It is possible that lysosomal membrane glycoproteins are somehow involved in the dynamics of lysosomes such as in the process of fusion of lysosomes with various other organelles.

It is interesting to compare some of the properties of human lamp-1 and lamp-2 presented here to those reported by others. For example, Lewis et al. (33) showed the presence of 18 N- glycosylation sites in rat lysosomal membrane glycoprotein(s) lgp 120 by endo-H digestion. They detected two protein bands on SDS-polyacrylamide gel electrophoresis after 20 h of digestion, and the ratio of these bands is approximately the same as seen here in Fig. 5, which represents lamp-1 and lamp-2. On -120-kDa glycoproteins of chicken and mouse cells, Lip- pincott-Schwartz and Fambrough (35) and Green et al. (40) showed a similar half-time for maturation of about 50 min and a similar doublet peptide after endo-H treatment. As shown in the following paper (30), the amino acid sequences of lamp-1 from human, mouse, and chicken are more than

50% identical. These results clearly indicate that lysosomal membrane glycoproteins in these different species are closely related to each other.

Our studies show that human lamp-1 and lamp-2 are major glycoproteins of the total cell mass. Although we did not attempt to isolate glycoproteins specifically from lysosomes, the glycoproteins obtained were derived from lysosomes. Sim- ilarly, Hughes and August (32) isolated mouse lamp-1 from total membranes. Additionally, lamp-1 and lamp-2 are major polylactosaminoglycan-carrying glycoproteins in various cells, and we have found them each to contain a significant amount of polylactosaminoglycan (Figs. 3, 8, and 11). These results led us to reevaluate some of the previous reports on polylactosaminoglycan-containing glycoproteins. For example, Den- nis et al. (50) reported that metastic cells contain more Glc- N A c P 1 4 branching on a-mannose than nonmetastatic cells. Since this branching is the most preferable site for polylactosamine extension (25, 51, 52), more polylactosamine is present in metastatic cells than nonmetastatic cells as a result. These structures mainly reside on Gp 130 in mouse cells and it is likely that Gp 130 is the mouse counterpart of lamp-1 or lamp-2 since both Gp 130 and lamps represent a major carrier for polylactosaminoglycan. It has been reported from several laboratories that tumor cells invade surrounding tissue or penetrate the endothelial membrane by secreting lysosomal enzymes (53-55). It will be intriguing to know how this increased secretion takes place in tumor cells and if any correlation exists between the secretion of lysosomal enzymes and surface expression of lysosomal membrane glycoproteins.

Acknowledgments-We thank Bonnie Kirstein and V. Shaub for technical assistance, Dr. I. J. Goldstein for the gift of Datura stramonium lectin, Dr. Torgny Stigbrand for help with monoclonal antibody production, Dr. Per-Ingvar Ohlsson for the amino acid se- quencing, Dr. Mark Williams for critical reading of the manuscript, D. Wey for preparing the photographs, and Tami Clevenger for secretarial assistance.

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SUPPLEMENTAL MATERIAL TO

h - L a w 1 AND h-lamp-2: MAJOR SIALOGLYCOPROTEINS CARRYING POLYLACTOSMIHOGL~CAN ISOLATION AND CHARACTERIZATION OF HUMN L I S O S M l A L MEMBRANE GLYCOPROlClNS

by

SYen R . CdrllPOn, Jurgen Roth. F r i e d r i c h P i l l e r and Minoru FUkuda

EXPERINWAL PROCEDURES

wyelogenOUS leukernla C e l l s (CHL) f o l l owed es~en t ia l l y t he p rocedure desc r ibed f o r 11oIation e; I euko r ia l i n f rom HL-60 Cells ( 1 4 ) . Packed CML c e l l s sere homgen i ted In I Y O ] . of 2% NP-IO. 150

was c e n t r i f u g e d f o I 20 mln. at 2OOOxg. TO the supernatant w a s added SOS and EDTA to f i n a l M NaCI, 1 M PI ISF, 50 nll TriP/HCl. pH 7.4. A f te r incubat ion on i re f o r 30 .in. the homogenate

was r e n t r l f u g e d f o r 1 h. at io0,ooorg and i h e __I_...I__... ".. I.I-.. ly

concent ra t ions 0.4% and 5 rd r e s p e c t i v e l y i

(1984) J. Biol. Chem. 259, 4792-4801

Bid. Chem. 259,11949-11957

J. Cell Bid. 1 0 5 , 1227-1240

39. Mizoguchi, A,, Takasaki, S., Maeda, S., and Kobata, A. (1984) J.

40. Green, S., Zimmer, K.-P., Griffiths, G., and Mellman, I. (1987)

41. Brown, W. J., Goodhouse, J., and Farauhar, M. G. (1986) J. Cell ~ i o i l 0 3 , i2x-1247

42. Griffiths. G.. Hoflack. B.. Simons. K.. Mellman, I., and Kornfeld, S. (1988) Cell 52, 329-341

. .

43. Kornfeld, S. (1986) J. Clin. Znuest. 77, 1-6 44. von Figura, S., and Hasilik, A. (1986) Annu. Reu. Biochem. 55,

167-193 45. Dahms, N. M., Lobel, P., Breitmeyer, J., Chirgwin, J. M., and

Kornfeld, S. (1987) Cell 5 0 , 181-192 46. Pohlmann, R., Nagel, G., Schmidt, B., Stein, M., Lorkowski, G.,

Krentler, C., Cully, J., Meyer, H. E., Grzeschick, K.-H., Mers- mann, G., Hasilik, A., and von Figura, K. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,5575-5579

47. Lobel, P., Dahms, N. M., and Kornfeld, S. (1988) J. Biol. Chern. 263,2563-2570

48. Oshima, A.. Nolan. C. M., Kyle. J. W.. Grubb, J. H., and Sly, W. s. (1988)'J. B i d Chem: 263; 2553-2562

49. Ho, M.-K., and Springer, T. A. (1983) J. Biol. Chem. 258, 636- 642

50. Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L., and Kerbel, R. S. (1987) Science 236, 582-585

51. Sasaki, H., Bothner, B., Dell, A,, and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076

52. Van den Eijnden, D. H., Koenderman, A. H. L., and Schiphorst, W. E. C. M. (1988) J. Biol. Chem. 263, 12461-12471

53. Oosta, G. M., Farreau, L. V., Bealer, P. C., and Rosenburg, R. D. (1982) J. Bid. Chem. 257, 11249-11255

54. Sloane, B. F., Rozhin, J., Johnson, K., Taylor, H., Crissman, J. D., and Honn, K. V. Proc. Natl. Acad. Sci. U. S. A. 83, 2483- 2487

55. Niedbala, M. J., Madiyalakan, R., Matta, K., Crickard, K.,

4641 Sharma, M., and Bernacki, R. J . (1987) Cancer Res. 47, 4634-

& v a t i o n o f q l v c o c w o t e i n r bv monoclonal antibody-Seoharose l f f l n i t v chromatoarauhl ~ The above mentloned glycoprotein fraction. whlch cantalned two Iyroromal membrane g lycopro te ins . vas used f o r production o f muse monoclonal ant lbod ie r ( 1 7 ) . Among several clones. one clone, which p roduced an t i bod ies w i th t he h ighes t a f f i n i t y aga ln r t one o f t h e g l y c a p r o t e i n r . was propagated and an t ibod ies were produced I S asc i tes ~n mice. This antibody. c a l l e d (03 (IgGl. kappa), was p u r i f i e d on protein A-Sepharore and coupled to Sepharore (4 w/ml g e l ) . The glycoproteins were parsed twice through the CUI-Sepharore m l u m w i t h 1 f low r a t e of 2 n l /h . A f te r each cyc le the column M I S washed w i t h 50 m1 o f 0.5% sodium deoxycholate. 10 nll l r i r / H C l . pH 8.0 and bound m a t e r i a l was eluted w i t h 50 n i l diethylamine. pH 12. E l u t e d f v a i t t o n s were ? m e d i a t e l y n e u t r a l - i z e d w i t h r a l i d g l y c i n e . The Separated glycoproteins were e r t e n r l v e l y d i a l y z e d a g a r n r t 6 1 s t ? l l c d water and lyophilized. The glycoprotein which dvd not bind to the c o l u m i s c a l l e d h . l m p - I . herea f te r , whereas the bound and e lu ted g l ycop ro te in i s ca l l ed h - l amp.2 .

Amino a c i d and carbohvdrate a$ described i n 1 P r e v i o u s r e p o r t I l k ) . The a n a l y r i r Of saccharides by Sephrder 6-50 and Bio-Gel

a n a l v r i r - h i n o a c i d and c a r b o h y d r a t e a n a l y l ~ r were c a r r i e d out

f-k gel f l l t n t i o n 1 1 s per fomed as descr ibed prev ious ly (20 21). Standard 0-l inked saccharides

obtained frem acute myelogenaur leukemia c o l l r b y endo-H d igor t ian o f g lycopept ider (unpub l ished were obta ined fmm 9ranu locy t l r Cells as descvibed 120) . St indard high-mannose saccharides were

r e s u l t s ) .


. The Icctln was used for th. detection O f N-acetyl- Iactoslmlnyl resl6ues (25.26) in I 110 step cytOChuIcal I f f l n l t y tcchnlquc (Egea and Roth. In

wlth the puvcl l u t l n (75 #+I, 15 .In) f o l l a d by rlnser r l t h PBS and lncubatlon prcparrtlon). Bre i f l y . u l t ra th ln srctlonr of L w l c r y l K4M nbc6ded HCLl cells =re Incubated

wlth ovaucold-gold complex (0.0. 525 nm - 0.25, 30 .In). Specl f lc l ty Of Iect ln rtalnlnq was controlled by prelncubatlon of the l e c t l n r l t h 0vD.uc0I6. I I I ~ ~ w ~ o o ~ o . u c O I ~ or N-aceytllac- tor r l ine. and by pletreatment o f the th in sectlons r l t h endo F ( 2 7 ) .

A B C D E F G H kDa

200-

116- 92.5-

66-

45-

A B 1 2 3 1 2 3

kDa

200-

116- 92.5-

66-

4 5-

A f R a

kDa

200-

92.5- 116-

06-

45-

0 0' 0' 10'20'30'45'60'90'120'120' - . . . * * . . . -

kDa kDa 200-

116- ' -125

92.5- -05

66-

45-

30-

20-

4 1 . 5 -38.5

1

3 0 1 0 Y l M 7 0 8 ) S Q I W l J

F~acIm

FIG. 12 p n 1m-1 md I-.

A. Sephadex G-50 gel f l l t r a t i o n Of alkallne borohydrlde-treated glyc0p.ptld.s r l t h hlgh mlecular welghts (Fnct lonr 28-40 I n Flg. 111). 51mIlw pro f l l es were obtained fm Other glycopeptlder r l t h hlgh nolecular welghts (Fractions 28-40 In Flp. 118. C and 0) .

8. 010-Gel P-4 gel f l l t n t l o n Of fractions 64-80 I n Flg. I2A. The 6 and 5 denote the elution posltlon of NevWlc~2-3G~ldl~GlcN4c~l~(NeuN4~2-3G~lpl-3)G~lN4cWl and Gt1414G1cWbl-6- (NeuNAc02-3Galbl-3) GaIHAcal.

C. Sephadex 6-50 gel f i l t r a t l o n Of glycowpt ldr k In Flg. 110 after mdo-b-l-ac8tylgluco- sulnldase H treatment. Man8 and MJn5 denote the elut lon porltims O f Ilan8GlcN4cWl and nln5- GlCN4CW.

FIG. 1 -h HL-60 cU.

A. Cells were labeled r l t h 3SS.methl~nin~ for IO .In. and chased the radioactivity r l t h vnlabeled r t h l o n l n e f a r lndlcated prlodr o f tlr. The l y so rou l &ram glycoprolelnr ere

gel elrctrophoreris ( 9 % Icrylamlde). As a control. the s u p l e taken after 120 .In of chase was l u n o p r e c l p l t a t e d by mtl.(h-Iamp-l + h - l up -2 ) ~ n t k d l e s and malyzed by SOS-polyxrylulde

~ m n o p r c c i p l t a t c d r l t h noma1 rabbl t scrv. (120' + NRS). 8. The Same sup les I S p ln r l A were treated wlth endo-H ( + ) before analysis. The cootroll were untreated ( - ) .

Polylactosaminoglycan-containing Lysosomal Membrane Glycoproteins T M L E I

UURIICTERISTICS OF Hlwl I M P - I NCJ LAW-2

l"1 l W . 2 T u p - 1 + law-2' l"l 1.9-2

llr Of nature glycoprotein

MI of precursor glycoprotein

Ir of endo-ll dlgerted precursor

N-terminal sequence

N m r o f N-glycans

Carhahydrate Cowosition. mler/nole

FUCOIe

llrnnare

G111ctase

GlcNAc

-ItOK

87.5N

39.5u

hNFllVKN616-

18

36.0

73.5

73.5

98.9

-1251:

84K

41.5K

LELXLTDSEX.

16

12.5

52.0

70.7

95.1

G a l M 15.4 8.2

Sialic acid 36.4 23.6

Percent carbohydrate 5%

10.6 8.6

10.4 6.8 4 .9 6.8 8.1 2.1 7.8 2.1 4.2 8.8 2.6 4 .4 2 . 1 4 .4 5.2

d

10.7 (Il.l)b 1.7 ( 8.7) 10.3 (10.3) 8.2 ( 6.7) 5.0 ( 4.9) 8.6 ( 8.2) 1.7 ( 2.1)

2.7 ( 2.1)

7.8 ( 6.7)

7.2 ( 7.7)

3.4 ( 4.1) 8.8 ( 9.0) 2.6 ( 2.6) 4.5 ( 4.6) 2.5 ( 2.1) 4.1 ( 4.6) 4.1 ( 4.4) - ( 0 . 3 )

13.8 (14.S)b 10.6 (10.6) 9.0 ( 8.2) 8.2 ( 7.1) 3.7 ( 1.9) 7.0 ( 5.3) 1.0 ( 6.8) 1.7 ( 1.1)

5.1 ( 5 . 0 ) 1.4 ( 1.3)

4.1 ( 4 . 1 )

7.2 ( 7.6)

7.8 ( 8.4)

18919

isolation and characterization of human lysosomal membrane ... · matteson, j., and carlsson, s. r....

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