(Received for publication, May 16, 1995; and in revised form, August 9, 1995)
From the
Mammalian cells express two different mannose 6-phosphate receptors (MPR 46 and MPR 300), which both mediate targeting of Man-6-P-containing lysosomal proteins to lysosomes. To assess the contribution of either and both MPRs to the transport of lysosomal proteins, fibroblasts were established from mouse embryos that were homozygous for disrupted alleles of either MPR 46 or MPR 300 or both MPRs. Fibroblasts missing both MPRs secreted most of the newly synthesized lysosomal proteins and were unable to maintain the catabolic function of lysosomes. The intracellular levels of lysosomal proteins decreased to <20%, and undigested material accumulated in the lysosomal compartment. Fibroblasts lacking either MPR exhibited only a partial missorting and maintained, in general, half-normal to normal levels of lysosomal proteins. The same species of lysosomal proteins were found in secretions of double MPR-deficient fibroblasts as in secretions of single MPR-deficient fibroblasts, but at different ratios. This clearly indicates that neither MPR has an exclusive affinity for one or several lysosomal proteins. Furthermore, neither MPR can substitute in vivo for the loss of the other. It is proposed that the heterogeneity of the Man-6-P recognition marker within a lysosomal protein and among different lysosomal proteins has necessitated the evolution of two MPRs with complementary binding properties to ensure an efficient targeting of lysosomal proteins.
In mammalian cells, mannose 6-phosphate receptors (MPRs) ()are essential elements of the transport system that ensure
the targeting of newly synthesized lysosomal enzymes. Soluble lysosomal
proteins become specifically modified with mannose 6-phosphate
(Man-6-P) residues during their passage through the Golgi apparatus and
are targeted by MPRs to lysosomes (for review, see Kornfeld(1992) and
Kornfeld and Mellman (1989)). The MPRs are type I transmembrane
glycoproteins and have apparent molecular masses of 46,000 Da (MPR 46)
(Hoflack and Kornfeld, 1985) and 300,000 Da (MPR 300) (Kornfeld, 1992).
The extracytoplasmic domain of MPR 46 is homologous to each of the 15
repeating units that build the extracytoplasmic domain of MPR 300. MPR
300 is known to bind and endocytose additionally nonglycosylated
insulin-like growth factor II (IGF-II). This property of MPR 300 is
considered to be of importance for controlling the extracellular IGF-II
level (Filson et al., 1993), while it is unclear whether MPR
300 has a function in transmembrane signal transduction analogous to
other growth factor receptors (Okamoto et al., 1990;
Körner et al., 1995).
The importance of MPRs for the biogenesis of lysosomes is illustrated by the phenotype of I-cell fibroblasts, which are unable to add Man-6-P recognition markers to their newly synthesized lysosomal proteins. As a result, excessive amounts of lysosomal proteins are secreted, and a marked intracellular deficiency of lysosomal enzymes and a defective lysosomal catabolism are observed (Nolan and Sly, 1989). As all mammalian cells analyzed express both MPRs, albeit at different levels and variable relative ratios (Wenk et al., 1991), it has been difficult to assess the role of each of the two MPRs in the biogenesis of lysosomes. Each of the two MPRs contributes to the intracellular retention of lysosomal proteins and their targeting to lysosomes as indicated by the accumulation of MPR ligands in the secretions of cells missing MPR 46 or MPR 300 (Gabel et al., 1983; Ludwig et al., 1993; Wang et al., 1994). It is, however, unclear whether the two MPRs transport the same or different lysosomal proteins or different subpopulations of a lysosomal protein, whether they feed distinct populations of target organelles with newly synthesized lysosomal proteins, and to what extent they can replace each other.
Answers to these questions would be greatly facilitated if cells could be studied that lack both MPRs and that allow the expression of either or both of the receptors at various levels. In this study, we report on the generation of mouse embryonic fibroblasts that lack MPR 46 and MPR 300. Mice strains carrying disrupted MPR 46 or disrupted MPR 300 alleles were crossed to obtain offspring homozygous for each of the disrupted MPR alleles. As deficiency of MPR 300 leads to death before birth (Wang et al., 1994; Lau et al., 1994), fibroblasts were established from embryos at day 12.5. The phenotype of these fibroblasts was compared with that of fibroblasts from embryos lacking either of the two MPRs and with the phenotype of control fibroblasts. Compared with fibroblasts with a deficiency of one of the MPRs, the missorting of soluble lysosomal proteins was much more pronounced in double MPR-deficient fibroblasts. Analysis of individual lysosomal enzymes and of the Man-6-P polypeptides accumulating in secretions indicated that lysosomal proteins bind to both MPRs, although with different affinities. As a result, the two MPRs transport distinct complements of lysosomal proteins that consist of the same components, but at different ratios.
Figure 1:
Genotype and
protein analysis of MPR 46 and MPR 300 in mouse embryonic fibroblasts. A and B, Southern blot analyses of genomic DNA
isolated from embryonic fibroblasts (10 µg) digested with BglII and EcoRI, respectively. The wild-type
4.2-kilobase pair (kb) fragment is replaced in the MPR
46 mice by the recombinant 5.4-kilobase pair
fragment. In the MPR 300
mice, the wild-type
5-kilobase pair fragment is replaced by the recombinant 3-kilobase pair
fragment. C and D, MPR 46 and MPR 300 expression,
respectively, in membranes of cultured day 12.5 mouse embryonic
fibroblasts analyzed on a Western blot.
In fibroblasts lacking both MPRs (MPR 46/MPR
300
), the activities of the five soluble lysosomal
enzymes were reduced to 7-21% of the control (Fig. 2). In
single MPR-deficient fibroblasts, this decrease was less pronounced
(39-81% in MPR 46
fibroblasts and 42-80%
in MPR 300
fibroblasts). In each of the MPR-deficient
fibroblast lines, the intracellular activity of acid phosphatase was in
the range of controls (95-107%).
Figure 2:
Intracellular activity of lysosomal
hydrolases in control and MPR-deficient mouse embryonic fibroblasts.
The MPR genotype of the cells is indicated at the top. The specific
activity is represented as shown: MPR 46/MPR
300
(&cjs2113;), MPR 46
/MPR
300
(&cjs2118;), MPR 46
/MPR
300
(&cjs2110;), and MPR 46
/MPR
300
(
). The data were collected from a single
fibroblast culture for each genotype. The assays were done in
duplicate; mean values are given.
For the five soluble
lysosomal enzymes, the fraction of activity that accumulated in the
secretions during a 24-h incubation period varied between 5 and 33%
(expressed as percentage of enzyme activity in cells and medium). This
fraction increased to 72-93% in MPR 46/MPR
300
fibroblasts (Fig. 3). A deficiency of MPR
46 increased the fraction of enzyme in the secretions only to a minor
extent (10-41%), while a deficiency of MPR 300 produced an
intermediate level of accumulation (35-70%). Lysosomal acid
phosphatase was not detectable in secretions of embryonic fibroblasts.
Figure 3: Activity of lysosomal hydrolases in secretions of control and MPR-deficient mouse embryonic fibroblasts. The activity of five lysosomal hydrolases (see Fig. 2) was determined in the media, and the cells of mouse embryonic fibroblasts were collected after a 24-h culturing period as described under ``Materials and Methods.'' The MPR genotype of the cells is indicated at the top. The bars give the activity measured in the secretions in percent of total activity (cells plus media) and are as described for Fig. 2. The data were collected from a single fibroblast culture for each genotype. The assays were done in duplicate; mean values are given.
In summary, these results suggest that lysosomal enzymes that are
normally transported via a MPR-dependent mechanism are only poorly
retained by MPR 46/MPR 300
fibroblasts and are mostly released into the secretions. In
single MPR-deficient fibroblasts, the missorting is less pronounced. It
should be noted that the loss of either MPR 46 or MPR 300 produces a
similar decrease in the intracellular level of the lysosomal enzymes
tested, while the accumulation in their secretions is much more
pronounced in MPR 300
fibroblasts.
Figure 4:
Biosynthesis of cathepsin D and
-glucuronidase in control and MPR-deficient fibroblasts. Cultured
fibroblasts were metabolically labeled with
[
S]methionine. Cathepsin D and
-glucuronidase were immunoprecipitated from identical amounts of
trichloroacetic acid-insoluble radioactivity of cells and corresponding
aliquots of the media and subjected to SDS-PAGE and fluorography. The
MPR genotype of the cells is indicated at top. Molecular mass standards
(in kilodaltons) are given on the left and right. M, mature
form; I, intermediate forms; P, precursor. The
relative level of newly synthesized cathepsin D and the percentage of
newly synthesized protein detectable in the secretions were calculated
by densitometric analysis and are given at the bottom. The asterisk indicates a reactive protein in the medium unrelated to cathepsin
D.
This clearly demonstrates that the bulk of newly synthesized
cathepsin D is secreted in MPR 46/MPR 300
fibroblasts, but also in MPR 300
fibroblasts.
Moreover, in cells lacking MPR 300 alone or in combination with MPR 46,
the relative level of newly synthesized cathepsin D was 2.3-2.4
higher than in control and MPR 46
fibroblasts.
Parallel to cathepsin D, -glucuronidase was immunoprecipitated
from cells and media.
-Glucuronidase is synthesized and secreted
as a 72-kDa precursor, while the intracellularly retained polypeptides
are processed to a 69-kDa mature form. In MPR 46
/MPR
300
fibroblasts, 86% of the labeled
-glucuronidase was recovered in the secretions in contrast to 6%
in controls. MPR 46
and MPR 300
fibroblasts accumulated in their secretions 10 and 69%,
respectively, of the labeled
-glucuronidase (Fig. 4),
indicating that missorting of
-glucuronidase and cathepsin D in
MPR-deficient fibroblasts is similar.
Figure 5: Immunofluorescence labeling of lamp-1 in normal and MPR-deficient mouse embryonic fibroblasts. MPR-deficient (B) and control fibroblasts (C) were analyzed by indirect immunofluorescence using anti-mouse lamp-1 antiserum. A light microscopic view of the MPR-deficient cells is shown in A. Magnification is 40-fold (A) and 63-fold (B and C).
Figure 6: Cathepsin D expression in normal and MPR-deficient fibroblasts. Homogenates (100 µg of protein) from cultured mouse embryonic fibroblasts were analyzed by Western blotting with a cathepsin D-specific polyclonal antiserum. The MPR genotype of the cells is indicated at the top. P, precursor; I, intermediate forms; M, mature form. The relative expression levels were measured by densitometry and are given below the lanes.
To determine the subcellular
location of cathepsin D, the postnuclear supernatants of control and
double receptor-deficient fibroblasts were subjected to Percoll density
gradient centrifugation (Fig. 7). This separates dense membranes
enriched in lysosomal markers (fraction 1) from more buoyant membranes
enriched in markers for the Golgi apparatus, plasma membrane, and
endosomes (fractions 4 and 5). In controls, 60-70% of the
lysosomal acid phosphatase and
-hexosaminidase was associated with
the dense and 15-20% with the light membranes (Fig. 7, A and B). In MPR 46
/MPR
300
fibroblasts, the fraction of both lysosomal
markers associated with dense membranes was reduced to 40-45%. In
control fibroblasts, the dense membranes contained the bulk of
cathepsin D (
80%) and essentially all of the 30-kDa mature form.
Similarly, in MPR 46
/MPR 300
fibroblasts, the dense membranes were enriched in proteolytically
processed forms, while the light membranes were enriched in precursor
forms. Thus, the residual amount of cathepsin D that is retained and
proteolytically processed in double receptor-deficient fibroblasts
appears to be associated with lysosomes.
Figure 7:
Subcellular fractionation of control and
MPR-deficient mouse embryonic fibroblasts by Percoll density
centrifugation. Postnuclear supernatants of cell homogenates were
subjected to Percoll density centrifugation. After fractionation and
removal of Percoll, the activity of lysosomal -hexosaminidase and
acid phosphatase was determined as described under ``Materials and
Methods.'' The activity is given as percent of total gradient
activity. Cathepsin D in the gradient fractions was analyzed by Western
blotting. P, I, and M denote the precursor
and intermediate and mature forms of cathepsin
D.
To obtain direct evidence
for a lysosomal localization of cathepsin D in MPR
46/MPR 300
fibroblasts,
cryosections were double-immunolabeled for lamp-1 and cathepsin D (Fig. 8). In control fibroblasts, the majority of cathepsin D
(small gold) and lamp-1 (large gold) label was associated with
lysosomal profiles that were filled with rather homogeneous
electron-dense material and multiple membranes. Profiles containing
only one type of label were rarely seen (Fig. 8A). This
was also true for MPR 46
/MPR 300
cells, in which two types of structures contained the bulk of the
label (Fig. 8B). One type resembled the lysosomal
profiles seen in controls, while the other type was represented by
large multivesicular structures filled with floccular electron-dense
material. Occasionally, mitochondria were detectable inside these
structures, suggesting an autophagic origin. Striking differences were
found for the relative intensities of cathepsin D and lamp-1 labeling
in the various structures (Table 1). In the lysosome-like
structures of controls, labeling for lamp-1 was
3 times higher
than for cathepsin D. In the autophagosome-like multivesicular
structures of MPR 46
/MPR 300
cells,
labeling for lamp-1 was 6 times higher, while in the lysosome-like
structures, labeling for cathepsin D was
6 times higher.
Surprisingly, the intensity of cathepsin D labeling seen in MPR
46
/MPR 300
fibroblasts did not
reflect the 5-10 times lower content of cathepsin D compared with
controls. Irrespective of the unexplained labeling intensities, these
results provide clear evidence that a small fraction of cathepsin D is
targeted to lysosomes in fibroblasts lacking MPR.
Figure 8:
Immunogold double labeling of cathepsin D
and lamp-1 in control and MPR-deficient mouse embryonic fibroblasts.
Cryosections of control and MPR 46/MPR
300
mouse embryonic fibroblasts were analyzed for
cathepsin D (10-nm gold) and lamp-1 (15-nm gold). A, in
control fibroblasts, both labels were found in lysosomal profiles
filled with rather homogeneous electron-dense material and multiple
membranes. B, in MPR 46
/MPR 300
fibroblasts, a second type of lysosomal profile was found that is
represented by large multivesicular structures (lower right part of B). Bar = 0.5
µm.
For this purpose, secretions of
metabolically labeled MPR 46/MPR 300
fibroblasts were passed over Affi-Gel 10 columns to which human
MPR 46 or MPR 300 had been coupled. After extensive washing, bound
ligands were eluted with Man-6-P and characterized by SDS-PAGE and
fluorography. Since the two MPRs were reported to have a distinct pH
dependence of binding above pH 6.5 (Hoflack et al., 1987;
Distler et al., 1991), we examined the binding over a pH range
of 5.4-7.4 (Fig. 9). Three aspects became apparent. First,
the pH profile for the overall binding of Man-6-P polypeptides showed
for both receptors an optimal binding between pH 6.2 and 6.6.
Rechromatography of the material unbound at pH 6.6 revealed that
>85% of the ligands had bound to either column during the first run.
Second, the pattern of polypeptides bound to the MPR 46 or MPR 300
affinity column differed, and the differences were observed over the
whole pH range tested. Receptor ligands with a pH optimum for binding
below pH 6.2 or above pH 6.6 were not apparent. Third, none of the
Man-6-P polypeptides bound exclusively to one type of MPR. Taken
together, these data indicate that the Man-6-P polypeptides differ in
their affinity for the two MPRs, but not to a degree that would prevent
binding to either of the two MPRs.
Figure 9:
pH-dependent binding of secreted
Man-6-P-containing polypeptides from MPR 46/MPR
300
mouse embryonic fibroblasts. MPR
46
/MPR 300
mouse embryonic
fibroblasts were labeled for 16 h with
[
S]methionine. The secretions were passed over
Affi-Gel 10 columns coupled with human MPR 46 (
) or MPR 300
(
) at the respective pH between pH 5.4 and 7.4. The fraction of
radioactivity that was bound and then eluted with 5 mM Man-6-P
was calculated (upper panel). The pattern of polypeptides in
the Man-6-P eluate was analyzed by SDS-PAGE and fluorography (lower
panel).
Due to the differential
affinities of Man-6-P polypeptides for either type of MPR, it was
expected that their pattern would be different in secretions from
either MPR 46 or MPR 300
fibroblasts. Secretions of MPR 46
cells were
expected to be enriched in Man-6-P polypeptides that bind
preferentially to MPR 46, and conversely, secretions of MPR
300
cells in those that bind preferentially to MPR
300.
The results shown in Fig. 10demonstrate that this
prediction was met only in part. While the frequency of Man-6-P
polypeptides that bound to the receptor columns increased from 1%
in controls to 3-4.5% in single MPR-deficient fibroblasts to up
to 7-8% in MPR 46
/MPR 300
fibroblasts, secretions from single MPR-deficient fibroblasts
contained Man-6-P polypeptides that bound almost equally well to either
type of receptor column.
Figure 10:
Binding of Man-6-P-containing
polypeptides in secretions of control and MPR-deficient mouse embryonic
fibroblasts to immobilized MPR 46 or MPR 300. Secretions of
metabolically labeled fibroblasts (genotype indicated at the top) were
passed over a MPR 46-Affi-Gel column (lanes A) or a MPR
300-Affi-Gel column (lanes B) at pH 6.6. The material bound to
the column and eluted with 5 mM M6P was separated by SDS-PAGE.
Molecular mass standards (in kilodaltons) are given on the left. The
numbers below the lanes represent the amount of bound S-labeled Man-6-P-containing polypeptides as a percent of
the trichloroacetic acid-insoluble
S-labeled material in
the secretions. The open arrowhead indicates the position of a
43-kDa polypeptide enriched in secretions of MPR 46
cells; the closed arrowhead indicates the position of a 68-kDa
polypeptide enriched in the secretions of MPR 300
cells.
Only few receptor ligands were markedly
enriched in secretions of either MPR 46 or MPR
300
fibroblasts, e.g. secretions of MPR
46
cells were enriched in a 43-kDa polypeptide.
Conversely, a 68-kDa polypeptide was enriched in secretions of MPR
300
cells. The 43- and 68-kDa polypeptides, however,
apparently bound equally well to the MPR 46 and MPR 300 affinity
columns (see Fig. 10). As predicted for the deficiency of both
MPRs, the 43- and 68-kDa polypeptides were also present in secretions
from MPR 46
/MPR 300
cells.
It
should be noted that the binding of Man-6-P polypeptides to MPR
affinity columns might significantly differ from the binding to MPRs in vivo. This is indicated by the observation that of the
cathepsin D polypeptides present in secretions of MPR
46/MPR 300
cells, only 26% were
bound to the MPR 46 affinity column and only 14% to the MPR 300
affinity column (data not shown). In vivo, the bulk of
cathepsin D is retained intracellularly as long as MPR 300 is
expressed, indicating efficient binding to MPR 300 (see Fig. 4).
Thus, the pattern of ligands identified by the affinity column approach
may significantly differ from the pattern of ligands that bind in
vivo to the MPRs in the Golgi/trans-Golgi network
compartment.
The deficiency of MPRs was predicted to result in a
similar missorting of soluble lysosomal proteins and lysosomal
dysfunction as the deficiency of Man-6-P recognition markers in I-cell
disease. In fact, a comparison of I-cell fibroblasts and MPR
46/MPR 300
fibroblasts shows that
this prediction is met. In both cell types, the bulk of newly
synthesized soluble lysosomal proteins is secreted. Intracellularly, a
profound deficiency of these proteins is found, and the lysosomal
compartment is enlarged due to storage material. These observations
underline the critical function of MPRs for targeting of soluble
lysosomal proteins. A similar conclusion was reached in a recent study
related to the present one, in which MPR-deficient fibroblasts were
studied that were obtained by mating MPR 46-deficient and T
mice (Ludwig et al., 1994). T
mice carry a
deletion on chromosome 17 that includes the locus of MPR 300. Since the
MPR 300 gene is imprinted in mice and only expressed from the maternal
chromosome (Wang et al., 1994), the maternal inheritance of a
T
allele results in a deficiency of MPR 300.
Biochemical and morphological data demonstrated that the cathepsin D retained in MPR-deficient cells was associated with lysosome-like structures. This raises the question by which mechanism the residual amount of lysosomal proteins found in lysosomes of MPR-deficient fibroblasts is transported to lysosomes. Signal receptor-dependent and signal receptor-independent (default) pathways, such as missorting at the trans-Golgi network, fluid-phase endocytosis, or cell-cell contact-mediated transfer of lysosomal proteins, may account for this residual targeting.
Support for transport of distinct complements of
lysosomal proteins by the two MPRs came from the comparison of the
Man-6-P polypeptides accumulating in secretions of MPR 46 and MPR 300
fibroblasts. Man-6-P polypeptides
preferentially accumulating in secretions of MPR 46
or MPR 300
fibroblasts were identifiable. This
clearly demonstrates that the two MPRs have distinct affinities for
Man-6-P polypeptides, which, in vivo and in vitro,
results in the binding of a different pattern of lysosomal proteins.
This was also supported by the distinct effect that loss of either MPR
had on the sorting of cathepsin D and
-glucuronidase. It should be
noted, however, that we failed to identify lysosomal proteins that
bound in vitro or in vivo exclusively to either type
of MPR.
Due to the overlapping affinities, it is conceivable that the two MPRs could substitute for each other with regard to their targeting function for lysosomal proteins provided that their expression level is high enough. There is already experimental evidence that overexpression of MPR 46 cannot compensate for the targeting function of MPR 300. In cells with undetectable levels of MPR 300, overexpression of MPR 46 led only to a partial correction of the missorting, while expression of MPR 300 in these cells fully corrected the missorting (Watanabe et al., 1990; Johnson and Kornfeld, 1992). This can be explained by the observation that part of the ligands that are bound by MPR 46 are secreted rather than targeted to lysosomes (Chao et al., 1990). Thus, the two receptors bind different complements of lysosomal enzymes and deliver them to sites that are different in part. Whether overexpression of MPR 300 can compensate for the loss of MPR 46 needs to be investigated.
If the two MPRs transport the same lysosomal proteins, although at different ratios, what parameter determines whether a lysosomal protein binds to MPR 46 or MPR 300? Available evidence suggests that lysosomal proteins encounter and bind to MPR 46 and MPR 300 in the trans-Golgi network (Klumperman et al., 1993). If so, structural differences in the Man-6-P recognition markers on lysosomal proteins must account for the binding of distinct complements of lysosomal proteins to the two MPRs. The Man-6-P recognition markers in lysosomal proteins indeed display a high structural heterogeneity. The number of phosphate groups in a high mannose oligosaccharide can vary (up to two), as well as the position of the phosphorylated mannose residues (five different positions) and the extent to which the phosphate groups persist in their biosynthetic precursor form as diesters (for review, see von Figura and Hasilik(1986)). Earlier studies have shown that the affinities of the two MPRs for high mannose oligosaccharides containing two phosphomonoester or phosphodiester groups differ significantly (Hoflack et al., 1987; Tong and Kornfeld, 1989).
Thus, the heterogeneity of Man-6-P recognition markers in combination with the inability of a single type of MPR to bind all the Man-6-P polypeptides with sufficient affinity to ensure their retention and targeting may have been one of the reasons that necessitated the evolution of two types of MPR. Theoretically, a higher efficiency of targeting may have also been obtained by adopting the N-acetylglucosamine 1-phosphotransferase to yield products of higher homogeneity and yet sufficient affinity for a single type of MPR. The N-acetylglucosamine 1-phosphotransferase, which is the key enzyme for the synthesis of Man-6-P recognition markers, has two recognition sites. Current models propose that one recognition site of the phosphotransferase interacts with an extended surface area of a lysosomal protein. The other recognition site then has to interact with oligosaccharides at multiple sites at the surface of a lysosomal protein on which the N-acetylglucosamine 1-phosphate residue is transferred (Cantor et al., 1992; Cantor and Kornfeld, 1992). It is conceivable that evolutionary pressure has focused on establishing the specificity of the N-acetylglucosamine 1-phosphotransferase for the heterogenous group of lysosomal proteins at the expense of the homogeneity of the Man-6-P recognition marker.
After having established two types of MPRs with complementary affinities for the diverse Man-6-P recognition markers, the two MPRs may have adopted additional functions, be it the endocytosis of Man-6-P ligands or the binding of IGF-II, both of which are characteristic of MPR 300. The acquisition of IGF-II binding appears to have been a fairly recent event in evolution since MPR 300 from chicken or frog fails to bind IGF-II (Canfield and Kornfeld, 1989; Clairmont and Czech, 1989).
Taken together, the studies on the single and double MPR-deficient fibroblasts led to the proposal that the heterogeneity of Man-6-P recognition markers necessitated more than one type of MPR to ensure efficient retention and targeting of the multiple forms of soluble lysosomal proteins. The heterogeneity of the Man-6-P markers may in turn have been the result of compromising the needs for distinguishing lysosomal from nonlysosomal glycoproteins by the N-acetylglucosamine 1-phosphotransferase and yet allowing phosphorylation of variably located oligosaccharides.