Lysosomal enzymes containing mannose 6‐phosphate recognition markers are sorted to lysosomes by mannose 6‐phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I‐cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6‐phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46‐deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 ‐/‐ mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 ‐/‐ mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase B (ASB), which is involved in degradation of dermatan sulfate and chondroitin 4-sulfate. A MATERIALS AND METHODSIsolation of a Genomic Clone and Targeting Vector Construction. A 758-bp partial murine ASB cDNA clone (pCABM750) was isolated from a Agtll brain cDNA library (Clontech) by homology screening with a full-length human ASB cDNA probe (4). The nucleotide sequence of pCABM750 was deposited in the GenBank/EMBL data base (accession no. X92096). pCABM750 exhibits 81.8% sequence identity with the human ASB cDNA (4) on nucleotide and 83.1% on deduced amino acid level (data not shown). In EcoRI digested genomic liver DNA of 129/SvJ mice a 9-kbp and a 1.6-kbp DNA fragment are hybridizing with pCABM750. A subgenomic DNA library was constructed by ligation of 8-to 10-kbp EcoRI DNA fragments from genomic liver DNA of 129/SvJ with X-EMBL4 arms and subsequent packaging according to the instructions of the supplier (Stratagene). A 9-kbp genomic clone (pmAB9E, see Fig. la) was isolated from the library by hybridization with pCABM750. For construction of a targeting vector the 6-kbp BamHI DNA fragment from pmAB9E was subcloned into pBlueskript SK-. A linker fragment from the BamHI site to the Sall site of the plasmid vector pUC18 was ligated to the XhoI site at the 5' end of the neomycin (neo) expression cassette of pMClneo poly(A) (ref.13; Stratagene); the neo cassette in pMClneo poly(A) contains a BamHI site at its 3' end. The neo cassette was inserted into the BglII site in exon 5 of the 6-kbp BamHI subclone described above as a BamHI-BamHI restriction fragment resulting in the targeting vector pAB6Bneo (see Fig. la). The insertion of the neo cassette introduces a premature translational stop codon

A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

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