GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain

1) Figure 2: the first experiment in the manuscript tests the effect of replacing phenylalanine residues in the TM span with leucine or alanine residues. The rationale for mutating these residues is not that clear since F, A and L residues are all typical amino acids for TM spans.

Our rationale was based on the relative hydrophobicity index of F, A and L. F and L have a similarly high hydrophobicity index whereas Ala has an ∼50% lower hydrophobicity index. We reasoned that if we changed 5 Phe residues to 5 Ala residues this would be a significant and potentially functional change, whereas changing to 5 Leu residues should have minimal effect, acting as a positive control. We have included this rationale in the text and added the lectin resistance data for Myc-GnT1IP/L(F/L) and Myc-GnT1IP-L(F/A) to revised Figure 2.

The lack of a wild type GnT1P-L control (Myc or HA tagged) in panel C is another weakness of this figure. The reader can't tell whether the (F/L) or (F/A) mutants are as effective as the parental construct. Oddly enough, the HA-tagged constructs are less effective than the Myc-tagged constructs. The authors don't comment on this tag-induced activity difference, as the next experiment (Figure 3) shows that both the cytosolic domain and the TM span of GnT1P-L can be replaced with the corresponding segments of MGAT1. Deletion of this figure would improve the manuscript since the results don't make a significant contribution after Figure 3 is shown. Figure 1 should be modified to eliminate the diagrams for the F/A and F/L constructs.

The L-PHA resistance test of a hygromycin-resistant transfectant population is not a directly quantitative test as it depends on the level of expression of a transgene in relation to hygromycin resistance. The most important parameter reflecting GnT1IP-L activity is whether a consistent proportion of transfectants survive high concentrations of L-PHA. Transfectant populations sorted for high GnT1IP-L expression or oligomannose expression show uniform resistance to L-PHA, as shown in our previous paper. We have now included an explanation in the text. Our previous paper also rigorously tested different tags on GnT1IP-L and found no consistent difference in HA versus Myc with respect to GnT1IP-L inhibitory activity. We have removed Figure 2 and altered Figure 1 as requested. However, we mention the results of the mutation experiment as the prelude to testing the GnT1IP-L luminal domain for inhibitory activity, and include some data in revised Figure 2.

2) Figure 3: the authors should explain the lectin growth inhibition assay better. Figure 3B would be improved by inclusion of wild type GnT1P-L and Lec1 cells. The main text needs to indicate that cell colonies are detected by staining with methylene blue. The text should also indicate that GnT1P-L expressing cells are not expected to show the same level of resistance as Lec1 cells, since MGAT1 activity is reduced, not eliminated.

We have expanded our description of the L-PHA resistance test and its interpretation as requested. We also included Lec1 in revised Figure 2. Since we did not test a wild-type control in every plate, we present a Myc-GnT1IP-L(F/L) control that was included in an experiment in which MGAT1/GnT1IP-L-Myc was also tested.

3) The authors should comment on the roughly 2-fold difference in observed FRET efficiency for the same donor/acceptor pair in Figure 5B versus Figure 5D. Is this explained by a difference in MGAT1 expression? The authors should consider changing the lettering for panel D to stress that the constructs tested (e.g., +MGAT1) are HA tagged, and are being tested as competitive inhibitors.

The lower FRET efficiency of mouse constructs likely reflects species differences as we observed lower expression levels of all mouse MGAT enzyme constructs compared to their human counterparts. The reason for this is unclear. However, if FRET efficiencies are expressed as a percentage of MGAT1/GnT1IP-L interaction, no differences are evident between mouse and human transferases. We have included a statement to this effect in the revised text. The lettering has been changed as requested in revised Figure 4.

4) Previous publications from Hassinen et al. have described Golgi-localized MGAT1-MGAT2 heteromers. The authors should test whether GnT1IP-L expression reduces MGAT1-MGAT2 heteromer formation using FRET and/or BiFC. This would provide insight into whether GnT1IP-L dissociates MGAT1 from MGAT2-MGAT1 heteromers, or instead exerts inhibitory activity within the context of pre-existing MGAT1-MGAT2 complexes. I believe the authors have the necessary cell lines and expression constructs to conduct this experiment.

The suggested experiments have been added to revised Figure 4 (panels E and F) and the figure legend modified accordingly. No differences were observed in MGAT1/MGAT2 interaction upon co-expression of a competing GnT1IP-L-HA construct with both human and mouse transferase constructs. This is consistent also with the inability of MGAT2-HA to inhibit MGAT1/GnT1IP-L interaction (see Figure 4D).

5) The final section of the manuscript makes an abrupt change to transcript profiling of GnT1IP-L and MGAT1 transcripts in mouse germ cells and male testis biopsies. This latter section of the manuscript was based on mining of previous microarray and RNA-Seq data sets for GnT1IP-L and MGAT1 transcript levels, but the data presentation and description of methods employed are cryptic or entirely missing. No descriptions of the informatics approaches for transcript profiling are presented in the Methods section and only literature references to the original data sets are listed in the figure legends. In addition, the descriptions in the figure legends and labeling of the figures (Figures 10 and 11) were incomplete or misleading. It is not clear from the figure or legend that Figure 10 is RNA-Seq data and the plot is labeled as “log2 fluorescence,” when it is likely supposed to be labeled “log 2 FPKM.” FPKM needs to be defined. A much more explicit description of the methods is required for Figure 10 and the axes need to be labeled in a comprehensible manner. The data in Figure 11 is also cryptically presented and it is not clear until the last line of the legend that the authors are describing a RNA microarray experiment. The connection between the transcript profiling and the remainder of the manuscript seems rather tenuous, but an effort is made to link protein expression with glycan phenotypes with glycan structure phenotypes in the respective cell types and human pathology of defects in spermatogenesis. This link from transcript levels to the glycan structures in the respective cell types needs to be more clearly presented so that it is clear what the take home message is for the gene expression data. Observations are made that GnT1IP-L expression is elevated in postmeiotic germ cells consistent with data in Figure 11 on human testis biopsies from men with impaired spermatogenesis. The authors hypothesize that blockage in glycan maturation is critical for germ cell interaction with Sertoli cells.

We have modified the text to introduce the context for presenting the gene profiling data and to include a take home message. We have also revised Figures 10 and 11 (now Figures 9 and 10) and their legends, added information to the Materials and methods section and rephrased the Results. Materials and methods now includes two sections: “Analysis of mouse RNA sequencing data” and “Analysis of human microarray data”, in which we describe in detail how datasets were downloaded, which Reads/Probesets were extracted, and how they were visualized with respect to germ cell subtypes and testicular phenotypes. The legends have been updated so that the legend title now contains data origin (RNA-Seq or Microarray). The x-axis of the histogram now has the correct label “log2 FPKM” (thanks for alerting us). Also FPKM is now defined in the legend to the new Figure 9 as well as in the Materials and methods section.

6) In the Discussion, the authors indicate that the previous paper (Huang et al., 2010) showed that the stem deletion mutant (delta-stem-GNT1IP-L) and the C-terminal deletion mutant of GNT1IP-L-CD1 still interact with MGAT1 but do not cause inhibition. For GNT1IP-L-CD1, Figure 4C of Huang et al. showed that the C-terminal deletion mutant is mainly retained in the ER, so formation of GNT1IP-L-CD1-MGAT1 heteromers should be reduced based upon the results presented in Figure 7 of the current manuscript. Unless I have overlooked something, interaction between a C-terminal GNT1IP deletion mutant and MGAT1 has only been tested in the context of the short form (GNT1IP-S-CD2 in Figure 5). Since the short form (GNT1IP-S) is not a MGAT1 inhibitor, one has to be concerned that ER retention of the truncation mutant is a contributing factor in lack of inhibitory activity. Since the current manuscript does not use BiFC or FRET to characterize formation of heteromers between MGAT1 and these inactive GNT1IP-L mutants, it seems premature to conclude that specific sub-regions of the lumenal domain are required for GNT1IP-L inhibitory activity.

We have added a description of the similarities and differences between GnT1IP-S and –L to the Introduction and elsewhere. Our previous paper showed that adding an N-terminal tag (Myc or HA) to GnT1IP-S converts it to a membrane-bound form that inhibits MGAT1 in transfected cells, indistinguishable from GnT1IP-L. The sequence of GnT1IP-L from aa 45 to 417 is identical to GnT1IP-S and differs only in that it has a 44 aa N-terminal extension. Some mutant constructs were made with Tag-GnT1IP-S which inhibits MGAT1 (Table 1, Huang and Stanley, 2010), and mislocalizes co-expressed MGAT1 to the ER more severely than Myc-GnT1IP-S-CD1 (C-terminal 39 aa deletion) (Figure 4B in Huang and Stanley, 2010). Tag-GnT1IP-S-CD1 does not induce resistance to L-PHA (Table 1), nor inhibit MGAT1 activity. Thus, it is appropriate to say that loss of the 39 C-terminal amino acids inactivates inhibition of MGAT1 by membrane-bound GnT1IP. Co-immunoprecipitation with MGAT1 was observed with a 121 aa C-terminal deletion, which suggests that the 39 aa deletion mutant would also interact with MGAT1. We have modified the revised text.