                         SEQUENCE LISTING

<110>  Rheinische Friedrich-Wilhelms-Universitt Bonn 
 
<120>  MUTATED ARYLSULFATASE A WITH INCREASED STABILITY

<130>  U31026WO

<150>  EP21157346.4
<151>  2021-02-16

<160>  8     

<170>  PatentIn version 3.5

<210>  1
<211>  507
<212>  PRT
<213>  Homo sapiens

<400>  1

Met Gly Ala Pro Arg Ser Leu Leu Leu Ala Leu Ala Ala Gly Leu Ala 
1               5                   10                  15      


Val Ala Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly 
            20                  25                  30          


Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn 
        35                  40                  45              


Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val 
    50                  55                  60                  


Pro Val Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg 
65                  70                  75                  80  


Leu Pro Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser 
                85                  90                  95      


Arg Gly Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala 
            100                 105                 110         


Ala Arg Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val 
        115                 120                 125             


Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe 
    130                 135                 140                 


Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr 
145                 150                 155                 160 


Cys Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu 
                165                 170                 175     


Val Pro Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro 
            180                 185                 190         


Trp Leu Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu 
        195                 200                 205             


Met Ala Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala 
    210                 215                 220                 


Ser His His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu 
225                 230                 235                 240 


Arg Ser Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala 
                245                 250                 255     


Ala Val Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu 
            260                 265                 270         


Glu Thr Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg 
        275                 280                 285             


Met Ser Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr 
    290                 295                 300                 


Thr Tyr Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly 
305                 310                 315                 320 


His Ile Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu 
                325                 330                 335     


Leu Pro Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr 
            340                 345                 350         


Leu Asp Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser 
        355                 360                 365             


Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg 
    370                 375                 380                 


Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr 
385                 390                 395                 400 


Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala 
                405                 410                 415     


Ser Ser Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser 
            420                 425                 430         


Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala 
        435                 440                 445             


Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala 
    450                 455                 460                 


Gln Leu Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly 
465                 470                 475                 480 


Glu Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg 
                485                 490                 495     


Pro Ala Cys Cys His Cys Pro Asp Pro His Ala 
            500                 505         


<210>  2
<211>  1524
<212>  DNA
<213>  Homo sapiens

<400>  2
atgggggcac cgcggtccct cctcctggcc ctggctgctg gcctggccgt tgcccgtccg       60

cccaacatcg tgctgatctt tgccgacgac ctcggctatg gggacctggg ctgctatggg      120

caccccagct ctaccactcc caacctggac cagctggcgg cgggagggct gcggttcaca      180

gacttctacg tgcctgtgtc tctgtgcaca ccctctaggg ccgccctcct gaccggccgg      240

ctcccggttc ggatgggcat gtaccctggc gtcctggtgc ccagctcccg ggggggcctg      300

cccctggagg aggtgaccgt ggccgaagtc ctggctgccc gaggctacct cacaggaatg      360

gccggcaagt ggcaccttgg ggtggggcct gagggggcct tcctgccccc ccatcagggc      420

ttccatcgat ttctaggcat cccgtactcc cacgaccagg gcccctgcca gaacctgacc      480

tgcttcccgc cggccactcc ttgcgacggt ggctgtgacc agggcctggt ccccatccca      540

ctgttggcca acctgtccgt ggaggcgcag cccccctggc tgcccggact agaggcccgc      600

tacatggctt tcgcccatga cctcatggcc gacgcccagc gccaggatcg ccccttcttc      660

ctgtactatg cctctcacca cacccactac cctcagttca gtgggcagag ctttgcagag      720

cgttcaggcc gcgggccatt tggggactcc ctgatggagc tggatgcagc tgtggggacc      780

ctgatgacag ccatagggga cctggggctg cttgaagaga cgctggtcat cttcactgca      840

gacaatggac ctgagaccat gcgtatgtcc cgaggcggct gctccggtct cttgcggtgt      900

ggaaagggaa cgacctacga gggcggtgtc cgagagcctg ccttggcctt ctggccaggt      960

catatcgctc ccggcgtgac ccacgagctg gccagctccc tggacctgct gcctaccctg     1020

gcagccctgg ctggggcccc actgcccaat gtcaccttgg atggctttga cctcagcccc     1080

ctgctgctgg gcacaggcaa gagccctcgg cagtctctct tcttctaccc gtcctaccca     1140

gacgaggtcc gtggggtttt tgctgtgcgg actggaaagt acaaggctca cttcttcacc     1200

cagggctctg cccacagtga taccactgca gaccctgcct gccacgcctc cagctctctg     1260

actgctcatg agcccccgct gctctatgac ctgtccaagg accctggtga gaactacaac     1320

ctgctggggg gtgtggccgg ggccacccca gaggtgctgc aagccctgaa acagcttcag     1380

ctgctcaagg cccagttaga cgcagctgtg accttcggcc ccagccaggt ggcccggggc     1440

gaggaccccg ccctgcagat ctgctgtcat cctggctgca ccccccgccc agcttgctgc     1500

cattgcccag atccccatgc ctga                                            1524


<210>  3
<211>  507
<212>  PRT
<213>  artificial

<220>
<223>  mutated human ARSA with increased enzymatic activity

<400>  3

Met Gly Ala Pro Arg Ser Leu Leu Leu Ala Leu Ala Ala Gly Leu Ala 
1               5                   10                  15      


Val Ala Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly 
            20                  25                  30          


Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn 
        35                  40                  45              


Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val 
    50                  55                  60                  


Pro Val Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg 
65                  70                  75                  80  


Leu Pro Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser 
                85                  90                  95      


Arg Gly Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala 
            100                 105                 110         


Ala Arg Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val 
        115                 120                 125             


Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe 
    130                 135                 140                 


Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr 
145                 150                 155                 160 


Cys Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu 
                165                 170                 175     


Val Pro Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro 
            180                 185                 190         


Trp Leu Pro Gly Leu Glu Ala Arg Tyr Val Ala Phe Ala His Asp Leu 
        195                 200                 205             


Met Ala Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala 
    210                 215                 220                 


Ser His His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu 
225                 230                 235                 240 


Arg Ser Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala 
                245                 250                 255     


Ala Val Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu 
            260                 265                 270         


Glu Thr Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Leu Met Arg 
        275                 280                 285             


Met Ser Asn Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr 
    290                 295                 300                 


Thr Tyr Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly 
305                 310                 315                 320 


His Ile Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu 
                325                 330                 335     


Leu Pro Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr 
            340                 345                 350         


Leu Asp Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser 
        355                 360                 365             


Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg 
    370                 375                 380                 


Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr 
385                 390                 395                 400 


Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala 
                405                 410                 415     


Ser Ser Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser 
            420                 425                 430         


Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala 
        435                 440                 445             


Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala 
    450                 455                 460                 


Gln Leu Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly 
465                 470                 475                 480 


Glu Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg 
                485                 490                 495     


Pro Ala Cys Cys His Cys Pro Asp Pro His Ala 
            500                 505         


<210>  4
<211>  507
<212>  PRT
<213>  artificial

<220>
<223>  mutated human ARSA with increased enzymatic activity and 
       stability


<220>
<221>  misc_feature
<222>  (424)..(424)
<223>  Xaa can be any naturally occurring amino acid

<400>  4

Met Gly Ala Pro Arg Ser Leu Leu Leu Ala Leu Ala Ala Gly Leu Ala 
1               5                   10                  15      


Val Ala Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly 
            20                  25                  30          


Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn 
        35                  40                  45              


Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val 
    50                  55                  60                  


Pro Val Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg 
65                  70                  75                  80  


Leu Pro Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser 
                85                  90                  95      


Arg Gly Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala 
            100                 105                 110         


Ala Arg Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val 
        115                 120                 125             


Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe 
    130                 135                 140                 


Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr 
145                 150                 155                 160 


Cys Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu 
                165                 170                 175     


Val Pro Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro 
            180                 185                 190         


Trp Leu Pro Gly Leu Glu Ala Arg Tyr Val Ala Phe Ala His Asp Leu 
        195                 200                 205             


Met Ala Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala 
    210                 215                 220                 


Ser His His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu 
225                 230                 235                 240 


Arg Ser Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala 
                245                 250                 255     


Ala Val Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu 
            260                 265                 270         


Glu Thr Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Leu Met Arg 
        275                 280                 285             


Met Ser Asn Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr 
    290                 295                 300                 


Thr Tyr Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly 
305                 310                 315                 320 


His Ile Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu 
                325                 330                 335     


Leu Pro Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr 
            340                 345                 350         


Leu Asp Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser 
        355                 360                 365             


Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg 
    370                 375                 380                 


Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr 
385                 390                 395                 400 


Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala 
                405                 410                 415     


Ser Ser Ser Leu Thr Ala His Xaa Pro Pro Leu Leu Tyr Asp Leu Ser 
            420                 425                 430         


Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala 
        435                 440                 445             


Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala 
    450                 455                 460                 


Gln Leu Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly 
465                 470                 475                 480 


Glu Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg 
                485                 490                 495     


Pro Ala Cys Cys His Cys Pro Asp Pro His Ala 
            500                 505         


<210>  5
<211>  538
<212>  PRT
<213>  artificial

<220>
<223>  mutated human ARSA with increased enzymatic activity and 
       stability and apoEII


<220>
<221>  misc_feature
<222>  (424)..(424)
<223>  Xaa can be any naturally occurring amino acid

<400>  5

Met Gly Ala Pro Arg Ser Leu Leu Leu Ala Leu Ala Ala Gly Leu Ala 
1               5                   10                  15      


Val Ala Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly 
            20                  25                  30          


Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn 
        35                  40                  45              


Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val 
    50                  55                  60                  


Pro Val Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg 
65                  70                  75                  80  


Leu Pro Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser 
                85                  90                  95      


Arg Gly Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala 
            100                 105                 110         


Ala Arg Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val 
        115                 120                 125             


Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe 
    130                 135                 140                 


Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr 
145                 150                 155                 160 


Cys Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu 
                165                 170                 175     


Val Pro Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro 
            180                 185                 190         


Trp Leu Pro Gly Leu Glu Ala Arg Tyr Val Ala Phe Ala His Asp Leu 
        195                 200                 205             


Met Ala Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala 
    210                 215                 220                 


Ser His His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu 
225                 230                 235                 240 


Arg Ser Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala 
                245                 250                 255     


Ala Val Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu 
            260                 265                 270         


Glu Thr Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Leu Met Arg 
        275                 280                 285             


Met Ser Asn Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr 
    290                 295                 300                 


Thr Tyr Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly 
305                 310                 315                 320 


His Ile Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu 
                325                 330                 335     


Leu Pro Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr 
            340                 345                 350         


Leu Asp Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser 
        355                 360                 365             


Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg 
    370                 375                 380                 


Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr 
385                 390                 395                 400 


Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala 
                405                 410                 415     


Ser Ser Ser Leu Thr Ala His Xaa Pro Pro Leu Leu Tyr Asp Leu Ser 
            420                 425                 430         


Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala 
        435                 440                 445             


Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala 
    450                 455                 460                 


Gln Leu Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly 
465                 470                 475                 480 


Glu Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg 
                485                 490                 495     


Pro Ala Cys Cys His Cys Pro Asp Pro His Ala Ser Ser Ala Trp Ser 
            500                 505                 510         


His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 
        515                 520                 525             


Ser Ala Trp Ser His Pro Gln Phe Glu Lys 
    530                 535             


<210>  6
<211>  30
<212>  PRT
<213>  artificial

<220>
<223>  apoEII tag

<400>  6

Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly 
1               5                   10                  15      


Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 
            20                  25                  30  


<210>  7
<211>  10
<212>  PRT
<213>  artificial

<220>
<223>  apoEII binding domain

<400>  7

Ser Ala Trp Ser His Pro Gln Phe Glu Lys 
1               5                   10  


<210>  8
<211>  10
<212>  PRT
<213>  artificial

<220>
<223>  linker

<400>  8

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 
1               5                   10  


