Information on EC 2.7.8.17 - UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase

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?

UDP-N-acetyl-D-glucosamine + cathepsin D

UMP + ?

5 entries

UDP-N-acetyl-D-glucosamine + DNase I

?

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-

?

UDP-N-acetyl-D-glucosamine + lysosomal-cathepsin Z D-mannose

UMP + lysosomal-cathepsin Z N-acetyl-D-glucosaminyl-phospho-D-mannose

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739732

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?

UDP-N-acetyl-D-glucosamine + lysosomal-enzyme D-mannose

UMP + lysosomal-enzyme N-acetyl-D-glucosaminyl-phospho-D-mannose

10 entries

UDP-N-acetyl-D-glucosamine + Man9GlcNAc1 oligosaccharide

?

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?

UDP-N-acetyl-D-glucosamine + methyl alpha-D-mannopyranoside

UMP + methyl (6-O-alpha-N-acetyl-D-glucosaminylphospho)-alpha-D-mannopyranoside

UDP-N-acetyl-D-glucosamine + methyl-alpha-D-mannoside

UMP + N-acetyl-D-glucosamine-phospho-(methyl-alpha-D-mannoside)

7 entries

UDP-N-acetyl-D-glucosamine + NPC2

?

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-

?

UDP-N-acetyl-D-glucosamine + ovalbumin

UMP + ?

UDP-N-acetyl-D-glucosamine + pro-tripeptidyl peptidase

?

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?

UDP-N-acetyl-D-glucosamine + ribonuclease B

UMP + ?

UDP-N-acetyl-D-glucosamine + RNase B

?

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?

UDP-N-acetyl-D-glucosamine + soybean agglutinin

?

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?

UDP-N-acetyl-D-glucosamine + thyroglobulin

UMP + ?

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weak activity

645370

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?

UDP-N-acetyl-D-glucosamine + UDP-glucose

UMP + ?

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?

UDP-N-acetyl-D-glucosamine + uteroferrin

?

3 entries

UDP-N-acetyl-D-glucosamine + uteroferrin

UMP + ?

additional information

?

-

12 entries

UDP-N-acetyl-D-glucosamine + lysosomal-cathepsin Z D-mannose

UMP + lysosomal-cathepsin Z N-acetyl-D-glucosaminyl-phospho-D-mannose

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739732

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?

UDP-N-acetyl-D-glucosamine + lysosomal-enzyme D-mannose

UMP + lysosomal-enzyme N-acetyl-D-glucosaminyl-phospho-D-mannose

9 entries

additional information

?

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Mg2+

Mn2+

(Man)5GlcNAc

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(Man)7GlcNAc

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ADP

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5 mM, 39% inhibition

ATP

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5 mM, 59% inhibition

cathepsin D

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deglycosylated cathepsin D inhibits the activity of both forms of GlcNAc-1-phosphotransferase toward cathepsin D

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EDTA

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10 mM, complete inhibition

methyl-alpha-D-mannoside

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NaCl

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0.1 M, 36% inhibition. 0.5 M, 91% inhibition

p-nitrophenyl-alpha-D-mannoside

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phosphatidic acid

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phosphatidylglycerol

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phosphatidylserine

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Sodium phosphate

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5 mM, pH 7.0, 55% inhibition

UDP

UDPGlcNAc

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UDPglucose

UTP

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5 mM, 34% inhibition

additional information

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D-glucose 6-phosphate

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activates

D-mannose 6-phosphate

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activates

Tergitol NP-10

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required

645369

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0.018 - 0.025

cathepsin D

3 entries

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0.03 - 0.06

DNase I

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3.3 - 5.7

Man9GlcNAc1 oligosaccharide

33 - 48

methyl alpha-D-mannopyranoside

0.021 - 183

methyl-alpha-D-mannoside

5 entries

0.352 - 0.367

NPC2

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0.11

pro-tripeptidyl peptidase

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0.916 - 1.244

ribonuclease B

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0.5 - 0.556

RNase B

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1

soybean agglutinin

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0.021 - 0.2

UDP-GlcNAc

5 entries

0.022 - 0.065

uteroferrin

4 entries

0.03 - 0.11

cathepsin D

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0.0045 - 0.005

DNase I

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0.75 - 1.55

Man9GlcNAc1 oligosaccharide

0.5 - 0.75

methyl alpha-D-mannopyranoside

0.44 - 0.62

NPC2

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0.008 - 0.038

pro-tripeptidyl peptidase

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0.042 - 0.05

RNase B

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0.0041 - 0.005

soybean agglutinin

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0.011 - 0.033

uteroferrin

1.2 - 4.45

cathepsin D

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0.08 - 0.15

DNase I

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0.23 - 0.266

Man9GlcNAc1 oligosaccharide

0.0166

methyl alpha-D-mannopyranoside

1.25 - 1.7

NPC2

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0.075 - 0.36

pro-tripeptidyl peptidase

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0.09

RNase B

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0.005

soybean agglutinin

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0.45 - 1

uteroferrin

0.733

UDPglucose

-

pH 7.6

0.00000038

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0.000733

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0.12

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6.82

after purification

6.5

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6.5 - 7.5

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with Tris-HCl giving 50% more activity than sodium cacodylate

6.6 - 7.5

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7

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7 - 8

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7.2 - 7.5

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7.4

5.5 - 8.5

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pH 5.5: about 50% of maximal activity, pH 8.5: about 75% of maximal activity

645376

6 - 9

Ceratitis capitata ARG-17

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about 50% of maximal activity at pH 6 and at pH 9

30

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37

20 - 40

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20°C: about 50% of maximal activity, 40°C: about 25% of maximal activity

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ARG-17 strain

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ARG-17 strain

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Dictyostelium discoideum

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UniProt

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135499, 645368, 645370, 645375, 645380

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peripheral blood mononuclear cell

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eye

high expression level

high expression level

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high expression level

761904

additional information

no activity in fibroblasts

739732

cis-Golgi network

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gamma subunit is localised in the cis-Golgi apparatus

5801

endoplasmic reticulum

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integral membrane protein

139

additional information

4 entries

malfunction

10 entries

physiological function

9 entries

additional information

4 entries

1000000

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145000

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166000

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2 * 166000 + 2 * 51000 + 2 * 56000, a 540000 Da enzyme complex is composed of disulfide-linked homodimers of 166000 and 51000 Da subunits and two identical, noncovalently associated 56000 Da subunits, SDS-PAGE

176000

SDS-PAGE

190000

228000

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enzyme from placenta, radiation inactivation

135495

270000

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alpha2beta2 GlcNAc-1-phosphotransferase

283000

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enzyme from skin fibroblasts, radiation inactivation

135495

330000

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alpha2beta2gamma2 GlcNAc-1-phosphotransferase

36000

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SDS-PAGE

41000

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45000

51000

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2 * 166000 + 2 * 51000 + 2 * 56000, a 540000 Da enzyme complex is composed of disulfide-linked homodimers of 166000 and 51000 Da subunits and two identical, noncovalently associated 56000 Da subunits, SDS-PAGE

56000

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2 * 166000 + 2 * 51000 + 2 * 56000, a 540000 Da enzyme complex is composed of disulfide-linked homodimers of 166000 and 51000 Da subunits and two identical, noncovalently associated 56000 Da subunits, SDS-PAGE

?

x * 190000, inactive mutant alphabeta precursor, SDS-PAGE, x * 45000, K4Q mutant beta subunit, SDS-PAGE, x * 41000, S15Y mutant beta subunit, SDS-PAGE

739540

dimer

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2 * 36000

heterodimer

heterohexamer

alpha2beta2gamma2

hexamer

additional information

8 entries

glycoprotein

7 entries

proteolytic modification

C447Y

Danio rerio

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site-directed mutagenesis, the mutation does not affect the catalytic activity of the GlcNAc-1-phosphotransferase. Enzyme-deficient embryos rescued with C447Y mRNA only corrects 84% of mutational features, and embryos rescued with the mutant mRNA appear visually different, i.e. shorter, from embryos rescued with wild-type mRNA

C473S

Danio rerio

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site-directed mutagenesis, the mutation does not affect the catalytic activity of the GlcNAc-1-phosphotransferase. Enzyme-deficient embryos rescued with C473S mRNA only corrects 80% of mutational features, and embryos rescued with the mutant mRNA appear visually different, i.e. shorter, from embryos rescued with wild-type mRNA

A34P

missense mutation of the alpha subunit leading to mucolipidosis II, mutation impairs retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes

A592T

site-directed mutagenesis, mutation involved in enzyme dysfunction, that has no effect on the mutant enzyme

A955V

site-directed mutagenesis, mutation involved in enzyme dysfunction

A955V/K928R

site-directed mutagenesis, mutation involved in enzyme dysfunction

C142S

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mutant expressed as dimer

C142Y

naturally occuring mutation of the gamma-subunit that causes misfolding of the gamma-subunit, the misfolded protein is retained in the endoplasmic reticulum, where it forms aggregates, and fails to rescue the lysososmal targeting of lysosomal acid glycosidases

C157S

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mutant expressed as dimer

C157S/C245S

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mutant can be detected as 36 kDa monomeric form but not as a dimer

C169S

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mutant expressed as dimer

C245S

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mutant can be detected as 36 kDa monomeric form but not as a dimer. Mutant is localised in the Golgi apparatus, indicating that dimerization is not essential for endoplasmic reticulum exit of the gamma-subunit. Monomeric C245S mutant does not assemble with endogenous GlcNAc-1-phosphotransferase 1 subunits

C442Y

site-directed mutagenesis, mutation involved in enzyme dysfunction, the Notch 1 mutant is efficiently delivered to the Golgi complex and the alphabeta precursor undergoes proteolytic cleavage

C461G

site-directed mutagenesis, mutation involved in enzyme dysfunction, the Notch 1 mutant is efficiently delivered to the Golgi complex and the alphabeta precursor undergoes proteolytic cleavage

C468S

site-directed mutagenesis, mutation involved in enzyme dysfunction, the Notch 1 mutant is efficiently delivered to the Golgi complex and the alphabeta precursor undergoes proteolytic cleavage

C505Y

C523R

site-directed mutagenesis, mutation involved in enzyme dysfunction

C84S

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mutant expressed as dimer

C84S/C157S

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mutant expressed as dimer

C84S/C157S/C245S

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mutant can be detected as 36 kDa monomeric form but not as a dimer

D1018G

site-directed mutagenesis, mutation involved in enzyme dysfunction

D190V

site-directed mutagenesis, mutation involved in enzyme dysfunction

D407A

site-directed mutagenesis, mutation involved in enzyme dysfunction, the mutant in the Stealth domain trafficks to the Golgi

D407A/A663G

site-directed mutagenesis, mutation involved in enzyme dysfunction

E36P

missense mutation of the alpha subunit leading to mucolipidosis III, mutation impairs retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes

F24V

missense mutation of the alpha subunit leading to mucolipidosis III, mutation impairs retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes

F374L

site-directed mutagenesis, mutation involved in enzyme dysfunction

G106S

naturally occuring mutation of the gamma-subunit that causes misfolding of the gamma-subunit, the misfolded protein is retained in the endoplasmic reticulum, where it forms aggregates, and fails to rescue the lysososmal targeting of lysosomal acid glycosidases

G126S

naturally occuring mutation of the gamma-subunit that causes misfolding of the gamma-subunit, the misfolded protein is retained in the endoplasmic reticulum, where it forms aggregates, and fails to rescue the lysososmal targeting of lysosomal acid glycosidases

G26D

missense mutation of the alpha subunit leading to mucolipidosis III, mutation impairs retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes

G575R

naturally occuring missense mutation identified in Brazilian mucolipidosis MLII/MLIII alpha/beta patients. The mutant shows 3% of wild-type activity

738351

H956Y

site-directed mutagenesis, mutation involved in enzyme dysfunction

I348L

site-directed mutagenesis, mutation involved in enzyme dysfunction, the mutant behaves similar to the wild-type enzyme

I403T

K1236A/R1237A/K1238A

mutant containing a mutation of C-terminal endoplasmic reticulum export motif mainly co-localizes with the cis-Golgi marker protein but fails to co-distribute with the endoplasmic reticulum protein marker

K1236M

K4Q

K732N

L1001P

site-directed mutagenesis, mutation involved in enzyme dysfunction, the mutant partially exits from the endoplasmic reticulum and is almost inactive

L1054V

site-directed mutagenesis, mutation involved in enzyme dysfunction

L5A/L6A

replacement of the N-terminal dileucine motif with alanine residues results in a significant reduction of the PT alpha/beta-subunit precursor protein cleavage. Densitometric evaluation of intensities of immunoreactive bands show that the formation of the beta-subunit is reduced by 46% compared with the wild-type construct. Mutant mainly co-localizes with the cis-Golgi marker protein but fails to co-distribute with the endoplasmic reticulum protein marker

L5A/L6A/R1253A/I1254A/R1255A

double mutant containing a mutation of N-terminal and C-terminal endoplasmic reticulum export motif shows a strong inhibitory effect on cleavage of the PT alpha/beta-subunit precursor protein. Mutation leads to retention in the endoplasmic reticulum

L5A/L6A/R1253L/I1254L/R1255X

the transfer of the dileucine motif to the C-terminal domain replacing the dibasic-based motif 1253RIR1255 in combination with the substitution of the N-terminal dileucine motif, blocks the endoplasmic exit and the subsequent proteolytic cleavage to mature PT beta-subunit

L5R/L6R/R1253A/I1254A/R1255A

substitution of the N-terminal L-Leu/L-Leu motif by dibasic-based motifs RIR or RR in combination with alanine substitution of the C-terminal motif prevents the alpha/beta-subunit precursor protein from reaching the Golgi apparatus for cleavage

L785W

site-directed mutagenesis, mutation involved in enzyme dysfunction, the DMAP interaction domain mutation has full activity toward alpha-MM but impaired ability to phosphorylate lysosomal acid hydrolases

N1153S

site-directed mutagenesis, mutation involved in enzyme dysfunction

N115Q

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electrophoretic migration similar to wild-type at 34 kDa, mutant sensitive to PNGase F treatment

N88Q

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electrophoretic migration similar to wild-type at 34 kDa, mutant sensitive to PNGase F treatment

N88Q/N115Q

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electrophoretic migration at 31 kDa, double mutant insensitive to PNGase F treatment, non-glycosylated polypeptide. Non-glycosylated gamma-subunits do not colocalize with the Golgi apparatus marker GM130

Q926P

site-directed mutagenesis, mutation involved in enzyme dysfunction

R1242A/R1243A/R1244A

by mutation of the arginine motif in beta subunit it is shown that this signal is not a functional endoplasmic reticulum retention signal

R1244A/R1245A/R1246A

mutant containing a mutation of C-terminal endoplasmic reticulum export motif mainly co-localizes with the cis-Golgi marker protein but fails to co-distribute with the endoplasmic reticulum protein marker

R1253A/I1254A/R1255A

mutant containing a mutation of C-terminal endoplasmic reticulum export motif mainly co-localizes with the cis-Golgi marker protein but fails to co-distribute with the endoplasmic reticulum protein marker

R334L

site-directed mutagenesis, mutation involved in enzyme dysfunction, the mutant fails to exit from the endoplasmic reticulum and is inactive

R334Q

site-directed mutagenesis, mutation involved in enzyme dysfunction, the mutant fails to exit from the endoplasmic reticulum and is inactive

R587P

site-directed mutagenesis, mutation involved in enzyme dysfunction, the R587P mutant shifts from a predominant endoplasmic reticulum localization to a predominant Golgi localization

R879A

mutation does not affect the normal processing of the full-length alpha/beta precursor at residue K928, but abolishes cleavage at Q882 for the Dictyostelium discoideum replacement mutant

R925A

R925A/R879A

mutations result in complete loss of beta subunit formation

761082

R986C

site-directed mutagenesis, mutation involved in enzyme dysfunction, the mutant in the Stealth domain trafficks to the Golgi

S15Y

naturally occurring mutation in the N-terminal cytoplasmic tail of the alpha-subunit leading to a mislocated enzyme, mistargeting of the mutant enzymes to lysosomes, where they are degraded, or to the cell surface and release into the medium, but the mutant enzyme shows 41% of wild-type activity. Half-life of the mutant is decreased compared to the wild-type enzyme

S399F

T1223del

naturally occurig heterozygous mutation identified in Brazilian mucolipidosis MLII/MLIII alpha/beta patients. The mutant T1223del is correctly transported and proteolytically cleaved into mature alpha- and beta-subunits exhibiting 85% of GlcNAc-1-phosphotransferase activity of the wild-type enzyme

738351

T286M

naturally occuring mutation of the gamma-subunit that does not alter the gamma-subunit function, the mutant variant enters the Golgi like the wild-type enzyme

T644M

naturally occuring heterozygous mutation identified in Brazilian mucolipidosis MLII/MLIII alpha/beta patients. The mutant T644M is correctly transported and proteolytically cleaved into mature alpha- and beta-subunits exhibiting 50% of GlcNAc-1-phosphotransferase activity of the wild-type enzyme

738351

V182D

site-directed mutagenesis, mutation involved in enzyme dysfunction

V182D/Q205P

site-directed mutagenesis, mutation involved in enzyme dysfunction

V27D

missense mutation of the alpha subunit leading to mucolipidosis II, mutation prevents the cotranslational insertion of the nascent GlcNAc-1-phosphotransferase polypeptide chain into the endoplasmic reticulum

V28D

missense mutation of the alpha alpha subunit leading to mucolipidosis II, mutation prevents the cotranslational insertion of the nascent GlcNAc-1-phosphotransferase polypeptide chain into the endoplasmic reticulum

W81L

site-directed mutagenesis, mutation involved in enzyme dysfunction, that has no effect on the mutant enzyme

DELTA1-47

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N-terminal region (residues 1-47) of Saccharomyces cerevisiae Alg14 localizes its green fluorescent protein fusion to the endoplasmic reticulum membrane. Coimmunoprecipitation demonstrates that the N-terminal region of Alg14 is required for direct interaction with Alg7

G163A/G165A

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mutant fails to rescue a loss of Alg14 function. Mutant is inactivated by loss of conserved G-loop

V131A/I135A/V139A/V143A/V146A/F150A

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a mutant in which six hydrophobic residues are replaced with L-Ala is able to rescue the loss of Alg14 function, indicating that the mutated hydrophobic residues do not have a deleterious effect on Alg14 activity. Growth of these cells is extremely slow at 30°C

V131I/I135L/V139I/V143I/V146I/F150L

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a mutant in which six hydrophobic residues are replaced with L-Ala is able to rescue the loss of Alg14 function

additional information

15 entries

20 - 22

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16 h, less than 20% loss of activity

37

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2 h, less than 20% loss of activity

50

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10 min, complete loss of activity

treatment with papain or phospholipase C from Bacillus cereus inactivates the enzyme in a time- and dose-dependent manner

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0°C, 2 months, less than 10% loss of activity

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645375

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HPC4-Sepharose column chromatography and DEAE-Sepharose column chromatography

partial

alpha2beta2gamma2 and alpha2beta2 GlcNAc-1-phosphotransferase are stably expressed in CHO-K1 cells (transmembrane segments are missing)

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cloning of a single cDNA and gene that encodes a precursor of the alpha- and beta-subunits of human GlcNAc-phosphotransferase (gene symbol, GNPTAB). Transfection of the alpha/beta-subunits precursor cDNA with or without cotransfection of the regulatory gamma-subunitc DNA results in an increase in GlcNAc-phosphotransferase activity on low molecular weight substrate. This demonstrates that the alpha/beta-subunits precursor cDNA encodes the catalytic domain of the enzyme

662418

expressed in 293-T cells

671199

expressed in CHO-K1 cells

674538

expressed in Escherichia coli

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expressed in Escherichia coli, COS-7 cells and HeLa cells

gamma subunit is over-expressed in COS-7 cells

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gene GNPTAB encodes the alpha- and beta-subunits of GlcNAc-1-phosphotransferase, low efficiency of GNPTAB mRNA expression due to its large size

Danio rerio

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gene GNPTAB encodes the alpha/beta-subunit of GlcNAc-1-phosphotransferase, recombinant expression of C-terminally V5/His-tagged wild-type and mutant enzymes in HEK293 cells

gene GNPTAB, the alphabeta precursors of wild-type, and mutant K4Q or S15Y enzymes with a C-terminal V5-tag are expressed in HEK-293 cells

739540

gene GNPTG encoding the gamma-subunit of the enzyme, transient expression of wild-type and mutant gamma-subunits in HeLa cells

gene GNTPAB encoding the alpha/beta-subunits, quantitative real-time PCR expression analysis, recombinant expression of nucleotides 8-3715

Mus musculus

Q69ZN6

739081

genes GNPTABG encode the alpha2beta2gamma2 enzyme, generation of generate alpha/beta and gamma bicistronic constructs, recombinant expression of HA-tagged wild-type and GNPTAB-/- and GNPTG-/- mutant enzymes in HeLa cells, recombinant expression of domain deletion mutants in HEK-293 cells

738704

overexpression of gamma-chain of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase in NIH-3T3 cells induces accumulation of macromolecules, formation of large cytoplasmic vacuoles and decrease of lysosomal enzymes in cells. Transient ectopic expression of the gamma subunit in endoplasmic reticulum induces lowered lysosomal enzyme activity in cells

Mus musculus

Q6S5C2

661819

plasmids encoding WT and K732N alpha/beta cDNA with C-terminal V5/His tags are generated and transfected into HEK-293 cells and monitoring of the proteolytic cleavage of the alpha/beta precursor by the appearance of the mature V5-tagged beta-subunit. Recombinant expression of wild-type and mutant GST-DMAP domain