THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1992 by The American Societyfor Biochemistry and Molecular Biology. Inc.
Val. 267, No. 14, Issue of May 15, pp. 970&9712,1992 Printed in U.S.A.
A Human Lysosomal ar-Mannosidase Specific for theCore of Complex Glycans’ (Received for publication, December 26,1991)
Rita De GasperiSQ,Peter F. Daniell, and ChristopherD. WarrenSII From the $Carbohydrate Unit, Lovett Laboratories, Harvard Medical School and Massachusetts General Hospital, Charkstown, Massachusetts 02129 and the llEunice Kennedy Shriver Center for Mental Retardation, Waltham, Massachusetts 02254
A novel lysosomal a-mannosidase, with unique subOur recent studieson the specificity of human, bovine, and strate specificity, has been partially purified fromhu- feline, lysosomal a-mannosidases toward natural substrates man spleen by chromatography through concanavalin derived from complex, hybrid, and high-mannose glycans (1, A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. 2), have shown that thedegradation proceeds in anonrandom This enzyme can catalyze the hydrolysis of only 1 fashion according to ordered and reproducible pathways which mannose residue, that which is a(1+6)-linkedto the8- are very similar in the various species and are largely deterlinked mannose in the core of N-linked glycans, as mined by the structure of the initial substrate.The lysosomal found in the oligosaccharides Mana(l+6)[Mana(l+3)] degradation of oligomannosyl glycans derived from N-linked Man@(1+4)GlcNAc and Mana( 1+6)Man@( 1+4) glycoproteins proceeds in a cooperative bidirectional manner, GlcNAc. The newly described a-mannosidase does not by the action of the enzymes glycosylasparaginase and endocatalyze thehydrolysis of mannose residues outside of at thereducing terminus (3), and the core, even if they are a(l+6)-linked, and is not N-acetyl-8-glucosaminidase exo-a-mannosidase at the nonreducing terminus. Because mannose in the core, which active on the other a-linked is (1+3)-linked. The narrow specificity of the novel complex glycans are the most abundant class of glycans in mannosidase contrasts sharply with thatof the major mammalian N-glycoproteins, their degradation is of special branched tetrasaccharide Mana(l-+ lysosomal a-mannosidase, which is able to catalyze theinterest. Thusthe G)[Mana(l+3)]Man@(l+4)GlcNAc (3a),’resulting from the degradation of oligosaccharides containingdiverse linkage and branching patterns of the mannose resi- sequential action on complex glycans of a-fucosidase, sialidues. Importantly, although the major mannosidase dase, @-galactosidase,and @-hexosaminidase,is the major readily catalyzes the hydrolysis of the core a(143)- substratepresented to human lysosomal a-mannosidase. linked mannose, it is poorly active towards the a(l+ Similarly the pentasaccharide Mana(1 4 )[Mana(l+3)] 6)-linked mannose, i.e. the verysame mannose residue Man@(14)GlcNAc@( 14)GlcNAc(3a’), representing the for which the newly characterized mannosidase is spe- complete core of N-linked glycans, is the major substrate cific. The novel enzyme is further differentiated from presented to bovine and feline a-mannosidases, because cattle the major lysosomal a-mannosidase by its inability to and catslack the lysosomal endo-N-acetyl-8-glucosaminidase catalyze the efficient hydrolysis of the synthetic sub- activity present in humans (3-5). Our studies on substrates strate p-nitrophenyl a-mannoside, and by the strong 3a and 3a’ indicated that, regardless of the species, lysosomal stimulation of its activity by Co2+and Zn2+.Similarly a-mannosidase had a pronounced preference for the hydrolto the major mannosidase, it is strongly inhibited by ysis of the a(l+3)-linked mannose residue, since the only swainsonineand l,4-dideoxy-l,4-imino-D-mannitol,digestion product observed was either the trisaccharide but not by deoxymannojirimycin. Mana( 1 4 ) M a n @ (1 4 ) G l c N A c (human enzyme, from The presence of this novel a-mannosidase activity in 1+ c/3( human tissues provides the best explanation, to date, 3a) or tetrasaccharide M a w ( 1 4 ) M a n @ ( 1 4 ) G l c N A 4)GlcNAc (bovine, feline enzymes, from 3a’), which were for the structures of the oligosaccharides stored in human a-mannosidosis. In thiscondition the major ly- resistant to furtherdegradation. For the bovine enzyme,these sosomal a-mannosidase activity is severely deficient, results were confirmed by the dramatic differences in kinetic but apparently thea( 1+6)-mannosidase is unaffected, parameters for the isomeric substrates Mana(l4)Manj3( 1+ so thatthe oligosaccharide structures reflect the 4)GlcNAc2and Mana(1+3)Man@(14)GlcNAc2(2)? These results were particularly intriguing for the human unique specificity of this enzyme. lysosomal a-mannosidase because they unexpectedly failed to explain the origin of the principal oligosaccharide accumulated andexcreted in human a-mannosidosis, disease a caused * This investigation was supported in part by Grants DK 40930, by an inherited deficiency of this enzyme. In patients with H D 16942, and SO7 RR 05486-28 from the National Institutes of this disease, the major urinary oligosaccharide,accounting for Health, and by a grant from the Emmanuel Deutsch Fund. The costs 66% of the total excreted material, is Mana(l+3)Manp(l+ of publication of this article were defrayed in part by the payment of 4)GlcNAc (2a) (6, 7). However, based on our results, if it is page charges. This article must therefore be hereby marked “aduer- assumed that the stored oligosaccharides originate from the tisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. Q Recipient of the Hulda Irene Duggan Investigator Award of the Arthritis Foundation. Present address: Dept. of Neurology, New York University School of Medicine, 550 1st Ave., RR 210, New York, NY 10016. 11 To whom correspondence should be addressed. Tel.:617-7263748.
Numbering of the oligosaccharides is based on the number of mannose residues. To distinguish isomeric structures, a, b, c, etc. were arbitrarily assigned according to a convention established in previous publications (see Refs. 1and 2). For structures corresponding to the numbers and explanation of the use of a prime, see Table I. R. De Gasperi and C . D. Warren, unpublished work.
9706
Human Lysosomal a(l-&)-Mannosidase activity of residual a-mannosidase, the most abundant oligosaccharide, derived from complex glycans, should instead have been Mana( 14)Man/3( 14)GlcNAc(2b). This suggests that another enzyme, with different substrate specificity, could have been responsible for generating the stored materials, The same conclusion had been reached in previous studies using normal and a-mannosidosisfibroblasts, cultured in the presence of the a-mannosidase inhibitor swainsonine (8-10). In these studies the diseased and normal cells both accumulated tetrasaccharide 3a, whereas in the absence of swainsonine the mannosidosis cells accumulated trisaccharide 2a,lacking the a(lA)-linkedmannose residue, as found in the urine of patients with a-mannosidosis. The existence of a n enzyme capable of cleaving the a(l-&)-linked mannose from 3a was, however, only postulated, and was not demonstrated in vitro. Recently, we have detected this a(1-6)mannosidase activity in human a-mannosidosis fibroblasts, as well asinhumancontrol fibroblasts (11)3and normal human spleen. In thispaper we report the partial purification from human spleen, and the characterization of this novel mannosidase activity, which was found to be specific for the a ( 1 4 ) - l i n k e d mannose residue in the core of N-linked glycans.
9707
TABLE I Structures and numbering of the oligosaccharides usedm substrates for the lysosomal a(l4)-mannosidase or as HPLC standards forthe analysis of the incubation products Substrates 2b' and 3a' are identical to 2b and 3a, respectively, except that they contain adi-N-acetylchitobiose residue at thereducing end instead of a single GlcNAc residue. - . -~
1
5. 2b
h 46
w 3c 4c
6a
MATERIALS ANDMETHODS
Human spleen was obtained through the National Disease Re0 search Interchange, Philadelphia, PA. Liver from a cat with amannosidosis was obtained from Dr. Joseph Alroy, Tufts University, Boston, MA; human a-mannosidosis urinewas from a patient a t the Eunice Kennedy Shriver Center for Mental Retardation, Waltham, MA; urine and pancreas from swainsonine-intoxicated sheep were obtained from Dr. Lynn F. James, United States Department of Agriculture, Poisonous Plants Research Laboratory, Logan, UT, DEAE-Sephadex A-50, ConA-Sepharose, and Sephacryl S-300 were obtained from Pharmacia LKB Biotechnology Inc., Piscataway, NJ; p-nitrophenyl a-mannoside and endo-8-N-acetylglucosaminidaseD from the areas of the integrated peaks (2). One unit of enzyme was from Sigma; Bio-Gel P-4 from Bio-Rad; deoxymannojirimycin and defined as theamount of enzyme which cleaved 1 nmol of 3a/h. The swainsonine from Genzyme, Boston, MA. 1,4-Dideoxy-1,4-imino-o- same assay was used to study the specificity of the enzyme towards mannitol was kindly provided by Dr. G. W. J. Fleet, Oxford Univer- other oligosaccharides. In these assays 3 units of enzyme were used sity. and theincubations carried out for time periods ranging from 3 to 16 Preparation of Oligosaccharides-For structures and numbering' of h depending upon the substrate under study. K,,, and Vmaxfor subthe oligosaccharides used in this study see Table I. Oligosaccharides strates 3 a , 2b, and 6 b were determined by the Lineweaver-Burke were isolated from human a-mannosidosis urine, a-mannosidosiscat double-reciprocal plot. Due to the labor intensive preparation of the liver, or urine or pancreas from swainsonine-intoxicated sheep, as samples for HPLC analysis, routine nonquantitative assays were previously described (12-14). Their purity was assessed by HPLC4 carried out using 2b as substrate and the incubation mixtures anaanalysis and was always greater than 95% (2). Oligosaccharides 3a, lyzed by TLC (1,2). 2b, and Sa were prepared by digestion of the corresponding oligosacPartiul Purification of the a(l+ti)-Mannosidase Activity-Human charide containing a di-N-acetylchitobiose residue a t the reducing spleen was homogenized with 5 volumes of cold distilled water. The end, with endo-8-N-acetylglucosaminidaseD or humanspleen endo- homogenate was centrifuged at 10,000 rpm for 30 min andthe B-N-acetylglucosaminidase, followed by purification by Bio-Gel P4 supernatant saved. The supernatantwas then brought to 30% satuchromatography (2). ration with solid ammonium sulfate and the precipitate discarded. Assay of a-Mannosidme Actiuities-The major lysosomal a-man- The 0-30% supernatant was then adjusted to 80% saturation with nosidase was assayed with p-nitrophenyl a-mannoside (2 mM) (15). solid ammonium sulfate and the precipitate collected by centrifugaThe a(14)-mannosidasewas assayed with its putative natural sub- tion. The precipitated proteins were then dissolved in 10 mM sodium strate oligosaccharide 3a as follows. Thirty nanomoles of 3a (final phosphate buffer, 0.1 mM CaC12, 0.1 mM MgC12,and 0.1 mM MnC12, concentration 0.5 mM) were incubated a t 37 "C with the experimen- 0.15 M NaCl (ConA buffer) and dialyzed against the same buffer (2). tally determined amount of enzyme in a final volume of 60 pl of 50 The material was then applied to a ConA-Sepharose column (1.5 X mM Na acetate buffer, pH 4.0, 1 mM CoC12. The incubation was 20 cm) and washed extensively with ConA buffer. The bound proteins stopped by addition of 1volume of ethanol and boiling for 3 min. The were then eluted with 0.5 M methyl a-mannoside in ConA buffer, samples were then centrifuged and the supernatants desalted and concentrated by ammonium sulfate precipitation, and dialyzed reduced with NaBH4 as described (2). The HPLC analyses of the against 50 mM sodium phosphate, pH 7.0 (post-ConA fraction). This reaction products were performed exactly as described in ourprevious fraction was found to contain the bulk of the various glycosidase paper (2). Product identification was based on comparison of the activities including the a(1-6)-mannosidaseactivity. The post-ConA retention times with those of authentic standardoligosaccharides as fraction was then applied to a DEAE-Sephadex A50 column (1.5 X well as by coinjection of the incubation mixtures with the above 30 cm) equilibrated with 50 mM Na phosphate buffer, pH 7.0. The standard oligosaccharides. The percent hydrolysis was calculated column was washed with the same buffer to elute the unbound material. Under these conditions the bulk of the major lysosomal aP. F. Daniel, J. E. Evans, R. De Gasperi, B. G. Winchester, and mannosidase activity was eluted in the unbound fraction (see Fig. C. D. Warren, unpublished work. (5). 1A) along with the lysosomal endo-8-N-acetylglucosaminidase The abbreviations used are: HPLC, high performance liquid chro- This fraction was saved and used as a source of major lysosomal amatography; Endo D, endo-8-N-acetylglucosaminidaseD; ConA, con- mannosidase activity. The column was then eluted with 50 mM canavalin A; DIM, 1,4-dideoxy-1,4-imino-D-mannitol; DMJ, deoxy- sodium citrate, pH 6.0. The a(l-$)-mannosidase was eluted with the mannojirimycin. bulk of the proteins partiallyoverlapping the more acidic form of the
'
Human Lysosomal cu(l-&)-Mannosidme
9708
major lysosomal a-mannosidase (see Fig. lA). Separation of the two activities was achieved by Sephacryl S-300 chromatography. The fraction containing a(l-&)-rnannosidase activity was applied to a Sephacryl S-300 column (1 X 90 cm) equilibrated with 50 mM Na phosphate buffer, pH 6.2. The fractions containing a(l-&)-specific activity were pooledand concentrated (Fig. 1B). Theenzyme fraction obtainedafter gel filtration was essentially devoid of the major lysosomal a-mannosidase activity and was used for all the subsequent characterization experiments. From 140 g of human spleen, 385 units, with a specific activity of 22 units/mg of protein, were prepared.
0
.
1 .o
RESULTS
By using a combination of ConA-Sepharose, DEAE-Sephadex, and Sephacryl S-300chromatography, we were able to isolate a novel a-mannosidase, the specificity of which, unlike that of the major lysosomal a-mannosidase, is limited to the hydrolysis of the mannose residue-linked a ( 1 4 ) to the core ,f3(1 4 ) - l i n k e dmannose of N-glycans. In order to differentiate this activity from the major lysosomal mannosidase, our assay employed oligosaccharide 3a as substrate and HPLC to analyze the products. Previous specificity studies on the major lysosomal a-mannosidase indicated that this enzyme, regardless of the species of origin, cleaved 3a to yield exclusively2b (1, 2)) while the activity described here yields exclusively 2a. Therefore, by using 3a as substrate and HPLCto analyze the products we were able to detect the new activity and differentiate itfrom the major lysosomal a-mannosidase, based on the pattern of digestion products. In the post-ConA fraction both products were present. However, after the DEAE-Sephadex and Sephacryl S-300 chromatographies we were able to obtain a fraction which yielded only 2a and was essentially free of the major lysosomala-mannosidase (see Fig. 1B). This also confirmed that the a ( 1 4 ) specific activity is indeed a separate protein from the major a-mannosidase enzyme. Properties of the a(14)-Manmsidase-The mannosidase activity was optimal at acidic pH (around pH 4.0) and was nearly absent at pH 7.0. The acidic pH optimum, along with the fact that it is retained by ConA-Sepharose, strongly suggests the lysosomal origin of the enzyme. The molecular weight was determined by gel filtration through Sephacryl S300 to be about 180,000, smaller than those reported for both forms of the major lysosomal a-mannosidase, which ranged from 260,000 to 300,000 (16). As summarized in Table11, the enzyme activity was greatly stimulated by both Co2+and Zn2+,unlike the major lysosomal a-mannosidase which is activated by Zn2+but inhibited by Co2+(1). Fig. 2 shows the HPLC analysis of the hydrolysis products of 3a,and the effect of Co2+and Zn2+on the a(l+ 6)-mannosidase. Since in our hands Co2+gave morereproducible results, and also because of its inhibitory effect on the major lysosomal a-mannosidase, the assays were routinely carried out in the presence of 1 mMCoC12. The a ( 1 4 ) mannosidase was completely inhibited by 10 p~ swainsonine, a potent inhibitor of lysosomal and Golgi I1 a-mannosidases (17, 18).It was also inhibited 70% by 50 pM 1,4-dideoxy-1,4imino-D-mannitol, which has also been shown to be a potent inhibitor oflysosomal a-mannosidasein vitro (19), but a weaker inhibitor than swainsonine in cultured macrophages (20). It was not affected by deoxymannojirimycin (40 p M ) which is an inhibitor of the Golgi I a-mannosidase activity (21,22). Substrate Specificity of the a(l-&)-Manmsidase-This study with natural substrates demonstrates that the enzyme is uniquely specific for the a ( l a ) - l i n k e d mannose residue linked to the B-mannosyl residue of the N-glycan core. Thus i t was able to catalyze removal of this single mannose residue from oligosaccharides 3a and 2b (see Table 111).No evidence forthe occurrence of alternative reactions, or for further
0.5
0.0 0
20 40 60 80 100 120 I 4 0 160 180 200
0.9 -
0.8
”
-2-2 a 0
. I
I
R
B
2 0.7 -p 8 0.6 --
m u
0.5-0.4 --
0.3
--
0.2 T
o.l 0.0
0
10
20
30
40
50
60
70
80
Fractbn N W k r
FIG. 1. Preparation of a(l+b)-mannosidase fromhuman spleen extract. A , DEAE-Sephadex A50 profile of the fraction obtained after ConA-Sepharose chromatography. The post-ConA fraction (210 mg of protein) was applied onto a DEAE-Sephadex column as described under “Materials and Methods.” The arrow indicates the beginning of the elution with 50 mM Na citrate buffer, pH 6.0, (0,AZm“,J. The major lysosomal a-mannosidase was assayed with p-nitrophenyl a-mannoside (V, A4wnm).The a(14)-rnannosidase was assayed by incubating the fractions with 2b followed by TLC analysis of the incubation mixtures (see under “Materials and Methods”). The fractions containing the a(l+)-mannosidase activity are indicated by the black bur. B , Sephacryl S-300 elution profile of the a(l-&)-mannosidasefraction obtained from DEAE-Sephadex A50. The fraction containing the a(1+6)-mannosidaseactivity from DEAE-Sephadex (175 mgof protein) was applied in three separate aliquots onto a Sephacryl S-300 column (1.5 X 90 cm) as described The activity of the major under “Materialsand Methods” (0,AZmnm). lysosomal a-mannosidase was assayed as described above (V, A m nm). The a(1+6)-mannosidasewas assayed as described above; the fractions containing this activity are indicated by the black bur.
hydrolysis of the digestion products, was obtained. A comparison of theHPLC profiles of the hydrolysis products of tetrasaccharide 3a by: ( a ) the a(14)-mannosidase and (b) the major lysosomal a-mannosidase (see Fig. 3) clearly shows how the specificities of these two enzymes differ (see Scheme 1).The hydrolysis of the trisaccharide 2b similarly yielded the expected disaccharide Manp(l4)GlcNAc (1) (data not shown). The newly described mannosidase was not able to catalyze any cleavage of the a(l+3)-linked mannose residue from trisaccharide 2a, a substrate very readily degraded by the major lysosomal a-mannosidase (1).However, the a(l+ 6)-mannosidase was able to catalyze the hydrolysis of pentasaccharide 4b and hexasaccharide Sb (see Table 111), again by cleavage of 1specific mannose residue, as shown by HPLC were determined for 3a, 2b, (see Fig. 4). The K,,, and VmaX and Sb. As summarized in Table 111, the lowest K,,, was for tetrasaccharide 3a,while the highest V,, was for trisaccharide 2b.The kinetic parameters show that thea-mannosidase
Human Lysosomal cY(14)-Mannosidase TABLE I1 Effect of ions and inhibitors on the activity of lysosomal 4 - 6 ) mannosidase Pentasaccharide 3a (0.5 mM) was incubated with 3 units of a(l+ 6)-mannosidase for 3 h in the presence and absence of the various cations and inhibitors. The analysis and quantitation of the products was performed by HPLC (see “Materials and Methods”).
9709
TABLE 111 Specificity of the lysosomal a(l4)-mannosidase toward oligomannosylglycans For key to schematic structures presented in this table, refer to Table I. ND, K,,, and Vmaxwere not determined for the substrate 4b.
vmoc
KJn
0
-(
Activity %
Control +1 mM CoC12 +1 mM ZnS04 +lo p~ swainsonine* +50 p M DIMb +40 p M DMJb
100 711 1400 0 30 100
A
1
a4
Ob
€4
+“ neo
“The +, -, and ne qualitatively indicate stimulation, inhibition, and no effect, respectively, of the same ions and inhibitors on the activity of the major human lysosomal a-mannosidase, as reported in the literature (1,and references cited therein). The control for these assays was the activity in the presence of coc12.
A
02 -0
A
ND
No
1.o
29
L
0
5
1
0
1
5
A
D
0
C
4
P
P
0
5
1
0
1
5
0
15
10
Tlme. mirm(er FIG. 2. HPLC analysis of the products fromthe digestion of tetrasaccharide 3a with a(l+6)-mannosidase. showing the effect of metal ions. The substrate was incubated with 3 units of enzyme for 3 h as described under “Materials and Methods.” Panel A, control; panel E , +1 mM CoC12;panel C, +1 mM ZnS04. After the incubations, the oligosaccharides were reduced with NaBH, and analyzed by HPLC as described (2). The oligosaccharides corresponding tothe peaks in the profiles are identified by the schematic formulas (see Table I for explanation). The chromatogram in panel D is included to show that the unidentified peak at 4.98 min shown in panels A, E, and C is not Manp(l+4)GlcNAc, but an unknown contaminant.
was not as active on hexasaccharide Sb,in which the a(l+ 3)-linked mannose is substituted by 2 a(1+2)-linked mannose residues, as itwas on 3a and 2b,suggesting that this substitution interferes with the binding and the hydrolysis of the substrate. Although pentasaccharide 4b was not available in amounts sufficient for kinetic studies, the results obtained
1
a 0
, 5
1
, 0
1
5
0
L )t !
5
1
0
1
6
0
5
M
E
m e . minuter
FIG. 3. HPLC analysis of the products from digestion of tetrasaccharide 3a with (A) 3 units of a(l+6)-mannosidase or ( B )with 0.12 units of the major lysosomal a-mannosidase (DEAE-Sephadex unadsorbed fraction, see “Materialsand Methods”), showing that the specificity is different. Incubations were carried out for 3 h and theproducts analyzed as described (2). The oligosaccharides corresponding to the peaks in the profiles are identified by schematic formulas, as in Fig. 2. The chromatogram in panel C shows the results of coinjection of the samples analyzed in panels A and E .
under standard conditions indicated that 4b was hydrolyzed at rates similar to 6b.Interestingly, the a(14)-mannosidase was not active on oligosaccharides which had been reduced to alditols by sodium borohydride, indicating that the reducing
9710
Human Lysosomal cu(l4)-Mannosidase added, the activity towards the synthetic substrate could be detected after prolonged incubation. The activity in the absence of the cation was very poor even at a substrate concentration of 10 mM, and therefore we preferred to always use the natural substrates3a or 2b for assaying the enzyme. DISCUSSION
%
2b
SCHEME 1. Degradation of the tetrasaccharide 3a by the
a(l+6)-mannosidase andby the major lysosomal a-mannosidase. A
0
C
P
m a . minutes
FIG. 4. HPLC analysis of the products fromdigestion of ( A ) hexasaccharide Sb, and ( B )tetrasaccharide 4b with a(1+6)mannosidase. The substrates were incubated with 3 units of enzyme for 16 h and the products analyzed by HPLC as described (2). As in Figs. 2 and 3, the products are identified by schematic formulas. The chromatogram in panel C shows the analysis of reference oligosaccharides 3c and 4c, isolated from human a-mannosidosis urine, as described (35).
terminal GlcNAc residue is part of the recognition site of the enzyme. When pentasaccharide Mana( 1-3) [Mana(1 4 ) I M a n p ( 1 4 ) G l c N A c @ ( 1 4 )GlcNAc (3a’) and tetrasaccharide Mana(l-&)Man~(l-4)GlcNAc@(l-4)GlcNAc (2b’) were employed as substratesinstead of 3a and 2b,HPLC analysis indicated that the a(l4)-mannosidase had probably very little activity towards these oligosaccharides, but this experiment was complicated by the unexpected presence of contaminating lysosomal endo-@-N-acetylglucosaminidaseactivity, the bulk of which had been recovered in the fraction unbound by DEAE-Sephadex (see “Discussion”). Importantly, the a(14)-mannosidase was also inactive toward hexasaccharide Sa, in which the core a ( l 4 ) - l i n k e d mannose is substituted by another a( 1 4 ) - l i n k e dmannose residue. This shows that the activity is restricted to the mannose that is part of the core, and that it will not hydrolyze other mannose residues, even when they are onthe correct branch and have the correct linkage type. Less surprisingly, the a( 1 4 ) - m a n n o s i dase did not hydrolyze any mannose residue from the typical high-mannose oligosaccharide Man9GlcNAc (9). The activity towards p-nitrophenyl a-mannosidewas also examined. Under the standard assay condition used to assay the major lysosomal a-mannosidase (substrate concentration 2 mM, no cation added), the enzyme had very little or no activity toward the synthetic substrate. However, when the substrate concentration was raised to 5-10 mM and C0C12 was
This study was stimulated by the need to explain the origin of the oligosaccharides that are stored in a-mannosidosis. Unexpectedly, our studies on the substrate specificity of the major human lysosomal a-mannosidase, which is deficient in a-mannosidosis, did not provide an explanation, but instead suggested the involvement of another enzyme with different specificity (1).Previous studies with cultured fibroblasts from a-mannosidosis patients hadindicated the existence of a different lysosomal a-mannosidase, which was not affected by the disease, and which was postulated (8-10) to be capable of hydrolyzing t h e a ( l 4 ) - l i n k e d mannose residue in the tetrasaccharide 3a. Other previous studies had characterized the residual activity in these patients as having highly increased K,,, for the synthetic substrates 4-methylumbelliferyl a-mannoside andp-nitrophenyl a-mannoside, lower thermal stability, and asbeing activated by Co2+and Zn2+(23-26). At the time it was not clear whether the “residual” activity found in the patients with a-mannosidosis was a mutant form of the major lysosomal a-mannosidase or a separate activity. We were able to initiate a search for a(la)-mannosidase activity because of the availability in our laboratory of the putative natural oligosaccharide substrate, 3a. Studies with a-mannosidosis fibroblasts (11)3confirmed that they indeed contained such an activity, although they apparently lacked any measurable activity towards p-nitrophenyl a-mannoside. When an extract from normal human fibroblasts was incubated with 3a,we found that beside 2b,the product expected from the action of the major lysosomal a-mannosidase, the isomeric trisaccharide 2a was also formed. This result suggested that the activity found in the a-mannosidosis fibroblasts was probably due to an a-mannosidase activity normally present in the lysosomes, but which hadnot been characterized owing toits poor activity toward synthetic substrates. In other words, this was an enzyme present in normal cells that had previously gone undetected,not an activity somehow induced as a result of the disease condition. Because the amount of enzyme activity extractable from fibroblasts was very limited, and since we now knew that it was present in normal cells, we searched for such activity in human spleen. In order to differentiate this novel activity from the major lysosomal a-mannosidase, our assay initially employed tetrasaccharide 3a as substrate and HPLC to analyze the products, because we knew that this would be an effective way to separate the two possible isomeric cleavage products, 2a and 2b. The a(l-&)-mannosidase activity was readily detected in a ConA-Sepharose-adsorbed fraction prepared from spleen aqueous extract because incubation of this fractionwith 3a gave both 2a and 2b. Chromatography through DEAE-Sephadex A-50 and Sephacryl S-300 then separated the major lysosomal a-mannosidase from the a( 16) activity, which under our usual conditions of assay was devoid of activity towards the synthetic substrate.In thisway, 385 units of a(14)-mannosidase, with a specific activity of 22 units/mg protein, was obtained from 140 g of spleen. This enzyme preparation was devoid of the major lysosomal amannosidase. In regard to previous observations of metal ion stimulations of residual a-mannosidase activity, it is particularly interesting that the a(l4)-mannosidasewas greatly stimulated by
Human Lysosomal a(l4)-Mannosidase
9711
a findingthat agrees both Co2+and Zn2+,in contrast to themajor a-mannosidase these compounds were poorly hydrolyzed, which is inhibited by Co2+,and modestly activated by Zn2+ with conclusions about a postulated a(l4)-mannosidasein (1, 27). Furthermore, the activity towards the synthetic sub- the previous study of the major lysosomal a-mannosidase (1). strate p-nitrophenyla-mannoside was verypoor.We did However, when purified endo-@-N-acetylglucosaminidase(5) observe the a(l4)-mannosidase activity, at substrate con- was included in the same incubation mixture, the result of centrations of 5-10 mM in thepresence of the metal ion, after what appeared to be a concerted action between the two prolonged incubations. Together, these results suggest that enzymes was seen. These experiments were hampered by the the a-mannosidase originally described as residual, which had difficulty of trying to remove residual trace amountsof highly high K,,, for the synthetic substrates and was activated by active endo-@-N-acetylglucosaminidasefrom the a-mannosiZn2+ and Co2+ (23-26), is in fact the a(14)-mannosidase dase preparation. Nevertheless, the results seem to indicate a functional relationship between a(14)-mannosidase and described here. The most striking property of this novel a-mannosidase is endo-@-N-acetylglucosaminidasein the degradation of the its unique specificity. It has notbeen previously characterized core of N-linked glycans. It is relevant to note that we have for two reasons: ( a ) its poor activity for synthetic substrates also detected the a(14)-mannosidase activity in rat spleen, and ( b ) its strict specificity for a narrow subclass of natural an animal species that also possesses lysosomal endo-@-Noligosaccharide substrates. The availability in our laboratory acetylglucosaminidase activity, whereas we failed to detect it in bovine and feline tissues, species which do not have the of a large number of oligomannosyl glycans, containing a variety of linkage and branching patterns, as well as of an endoglycosidase activity (3, 4). The specificity of the a-mannosidase described herein proassay based on an HPLC system capable of separating structural isomers, greatly facilitated the detection and the char- vides, for the first time, a convincing explanation for the acterization of this novel a-mannosidase. Our specificity stud- origin of the major oligosaccharide stored and excreted in ies show that the activity of this enzyme is directed towards human a-mannosidosis. This storage product, trisaccharide 1mannose residue only, this being the one a(l-&)-linked to 2a, is derived from tetrasaccharide 3a by the action of the the @-linkedmannose of the N-glycoprotein core. It did not a(l-@-mannosidase. Tetrasaccharide 3a is in turn derived catalyze the hydrolysis of other (l&)-linked mannose resi- from complex glycans, by the action of several exoglycosidases dues if they were not in thecore, and it was inactive towards and lysosomal
[email protected] the the other core mannose residue that is linked a(1+3). Impor- a(l+6)-mannosidase cannot further hydrolyze 2a,this comtantly, if the core a ( l 4 ) - l i n k e d residue was substituted by pound accumulates in the absence of the major lysosomal aother mannose residues it was not active, i.e. it is not an mannosidase. Because complex glycans are the most abunendomannosidase. Thus of all the potential substrates tried, dant, comprising two-thirds or more of the totalN-glycans in two, 3a and 2b, were hydrolyzed rapidly, while two others, a given cell (28, 29), the end result is the massive storage of 4 b and 5b, in which the core is substituted by additional 2a. By similar logic, cats with a-mannosidosis, lacking both a(l+6)-mannosidaseand endo-@-N-acetylglucosaminidase, mannose residues on the a(1-3) branch, werehydrolyzed store instead the pentasaccharide Mana(1+3) [Mana(1-45)] more slowly. The kinetic parameters indicated that the enzyme has the highest affinity for the branched tetrasaccharide Manp(l4)GlcNAc@(14)GlcNAc(3a') in most tissues (12, 3a,although it hydrolyzed trisaccharide 2b, which lacks the 30, 31). Oligosaccharides 3c and 4c, which contain 1 or 2 nonrea(l+3)-linked mannose residue, more rapidly. It would have been interesting to extend the study to tetrasaccharide ducing terminal a(1+2)-linked mannose residues, respecMana( 1 4 ) M a n a ( l 4 ) M a n @ ( 1 4 ) G l c N However, Ac. this tively, have always posed an enigma for investigators of compound is not naturallyoccurring, and although the closely humana-mannosidosis. Although theseare major storage 1+ materials (6,7), together accounting for some 20% of the total related pentasaccharide Mana( 1+6)Mana( 14)Man@( 4)GlcNAc@(14)GlcNAcis found in the urine of swainson- oligosaccharides, their origin cannot be easily explained on ine-poisoned sheep (13), it is difficult to separate from two the basis of any known pathways for either the processing or isomeric pentasaccharides. Furthermore, the conversion of the breakdown of N-linked glycans. Previously, a hypothesis has been advanced (10) that proposes the addition of a(l+ Mana(l+6)Mana(l+6)Man@( l-A)GlcNAcp( 14)GlcNAc into the required tetrasaccharide can only be achieved by 2)-linked mannose residues to 3a to form first 3c and then 4c,provoked by the accumulation of 3a at nonphysiological digestion with human lysosomal endo-N-acetyl-8-glucosalevels.Analogous unorthodox glycosylation products have minidase, which presents additional problems. For these reabeen reported from patients with aspartylglucosaminuria (33) sons, M a n 4 1 4 ) M a n a ( 1 4 ) M a n @ ( 1 4 ) G l cwas N A cnot and @-mannosidosis(34). However, from this study, we now included in the study. In any case, the results for substrates know that 3c and 4c can be formed from 4b and 5b,respec2b, 3a,and Sa suggest that itwould not have been degraded tively, by the action of the a(l4)-mannosidase. Furtherby a(l+6)-mannosidase. Thus 5a, which can be viewed as more, from our recent studies on the specificity of cytosolic Mana(1+6)Mana(l-45)Man@(14)GlcNAc substituted by 2 a-mannosidases? we know that hexasaccharide 5b is the end cu(l+3)-linked mannose residues, was not degraded, and the product of the degradation in vitro of certain high-mannose resistance of this compound to theaction of a(l-&)-rnannos- glycans by human cytosolic a-mannosidase. Since some other idase is unlikely to have been due to the (1+3)-linked resi- high-mannose glycans, like 4b,contain only 1a(l+2)-linked dues, because the similar residue in 3a did not interfere with mannose residue on the (1+3) branch, it is probable that 4b its breakdown. Other results indicate that restriction of spec- can be formed similarly to 5b. The function of cytosolic aificity of a(14)-mannosidase extends to theN-acetylglucos- mannosidase is uncertain, but a role in oligomannosyl glycan amine-containing part of the substrate molecule. Thus, re- catabolism has been proposed (32). According to theproposed duction of the terminal GlcNAc residue rendered 3a resistant mechanism, cytosolic a-mannosidase is responsible for feedto theaction of the enzyme, suggesting that this residue must ing partially degraded substrates to lysosomes. Thus different be in the pyranose ring form for the substrate to be accom- high-mannose glycans would be converted into 5b and 4b by modated by the catalytic mechanism. Preliminary experiments with substrates containing a terminal di-N-acetylchiS. A1 Daher, R. De Gasperi, P. F. Daniel, S. Hirani, C. D. Warren, tobiose residue instead of a single GlcNAc indicated that and B. G. Winchester, Biochern. J.,in press.
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Human Lysosomal cu(l4hMannosidase
cytosolic a-mannosidase, and further breakdown of 6 b and 4 b would occur bythe action of lysosomal a-mannosidase. In the mannosidosis patients, the only lysosomal activity available is the a(1+6)-mannosidase, so 6 b and 4 b would end up as 4c and 3c,respectively. The concerted function of cytosolic mannosidase and a(l4)-mannosidase therefore offers a feasible explanation for the storage of 3c and 4c in human amannosidosis. Thus the characterization of the novel mannosidase presented here helps to explain the origin of the oligosaccharides stored in human a-mannosidosis. The results described in this paper, along with our previous studies on the specificities of the major lysosomala-mannosidase in humans, cattle, and cats (1, 2), explain why the structures of the major stored oligosaccharides are species specific, although the specificity of the major lysosomal a-mannosidase isessentially the same in the three species. It is now clear that the nature of the stored oligosaccharides reflects not only the presence or absence of significant levels of residual major a-mannosidase activity, but also the presence of other hydrolytic activities. The advantage gained by human tissues in possessing this additional a-mannosidase, with specificity for a single mannosidic linkage, is unclear, but itmay berelevant to note that the core a(14)-mannosidic linkage is very poorly handled by the major lysosomal a-mannosidase (1).It is possible that the a(l-&)-mannosidase serves as an accessory, albeit important activity, that has evolved to aid inthe efficient breakdown of complex glycans. Future studies on this novel a-mannosidase will be necessary to clarify its species distribution, and to shed light on its relationship with the major lysosomal a-mannosidase andwith lysosomal endo-N-acetylP-D-glucosaminidase, and to define its precise role in the catabolism of N-linked glycoproteins. Acknowledgments-We thank the National Disease Research Interchange for supplying human spleen, Dr. Joseph Ahoy for supplying a-mannosidosis cat liver, Dr. Lynn James for supplying urine and pancreases from swainsonine-poisoned sheep, and Dr. George Fleet for supplying 1,4-dideoxy-1,4-imino-o”annitol. We also wish to thank Dr. Roger W. Jeanloz for his advice and support.
1. 2. 3. 4. 5. 6. 7.
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