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Fig. 6. Multiple sequence alignments and annotation of Frem1 and related proteins. Alignments were generated by using ClustalW (1). Sequence identifiers are of the form species abbreviation (Mm, Mus musculus; Lv, Lytechinus variegatus; Ce, Caenorhadbitis elegans; Dm, Drosophila melanogaster; Fr, Fugu rubripes) followed by sequence accession number, if available, or gene name (see Table 1 for the origin of these sequences), followed by the amino acid coordinates for the aligned fragment of sequence where appropriate. (A) Alignment of Frem1 orthologous sequences. Consensus attachment sites for glycosylaminoglycan modification (including chondroitin sulfate attachment) are shown by residues, colored blue, and potential N-glycosylation sites, indicated in red. Consensus protein motifs were defined by the regular expressions "[ED].{0,3}SG" for glycosaminoglycan (2, 3) and "N[ˆP][ST]" for N-glycosylation (4). (B) Alignment of Frem2 and Frem3 amino acid sequences, annotated for glycosylaminoglycan and N-glycosylation as for A. (C) The alignment was colored by using the default scheme from chroma (5) based on a threshold of 60% conservation. Red boxes below the alignment indicate residue positions that have been shown to coordinate Ca²⁺ ions in the solved structures of cadherins (see C) and that are often conserved between chondroitin sulfate proteoglycan element (CSPG) domains and cadherins. Gray boxes indicate Ca²⁺ coordination residues that are not typically conserved between CSPG domains and cadherins. Green boxes indicate conserved negatively charged residues, located in regions of the CSPG domain that suggest they may be involved in Ca²⁺ coordination. (D) Multiple sequence alignment of cadherin domains for which crystal structures have been solved. Sequence identifiers are Swiss-Prot identifiers (6) that correspond to the full sequence of the solved structures. The partial structures of CAD1_MOUSE (mouse N-cadherin), CAD2_MOUSE (mouse E-cadherin), and CADF_XENLA (Xenopus laevis C-cadherin) are accessioned under the Protein Data Bank (PDB) as PDB ID codes 1NCJ,1EDH, and1L3W, respectively (7-9). This alignment is colored with the default scheme from chroma as if it were aligned with the sequences in C to highlight both consistencies and divergence between these related domains. (E) Secondary structure assignments [Database of Secondary Structure of Proteins (10)] for the alignment shown in B. b -Strand residues are indicated by E and highlighted in bold. Residues directly coordinating Ca²⁺ ions in the solved structures are highlighted with a red background. Residues that were not resolved in the structures are indicated by ?. Ca²⁺ ions are intercalated between consecutive cadherin domains in these solved structures; consequently, terminal domains will have less Ca²⁺ contact residues. N-terminal domains are indicated by # and C-terminal domains by $ after the sequence coordinates; the remaining domains are not terminal.

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